Schiff bases and the metal(II) complexes were established by elemental analyses
. ... Four unsymmetrical and five symmetrical Schiff base complexes of.
METAL(II) SCHIFF BASE COMPLEXES AND THE INSULIN-MIMETIC STUDIES ON THE OXOVANADIUM(IV) COMPLEXES
BY
ADEOLA AYODEJI NEJO 206001421 B. Sc. (Hons), M. Sc. (Lagos)
A THESIS IN THE DEPARTMENT OF CHEMISTRY
SUBMITTED TO THE FACULTY OF SCIENCE AND AGRICULTURE IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF
DOCTOR OF PHILOSOPHY OF THE UNIVERSITY OF ZULULAND
JUNE 2009
ABSTRACT
Sixteen symmetrical and four unsymmetrical tetradentate Schiff bases with the N2O2 chromophore were isolated in pure form and fully characterized by elemental analyses, melting point, IR and 1H NMR. The appearance of two different peaks for each of the azomethine protons and phenolic protons confirm the asymmetry nature of the unsymmetrical Schiff bases. All the Schiff bases were successfully coordinated to oxovanadium(IV) ion to form the corresponding complexes. The unsymmetrical Schiff bases were also successfully coordinated to cobalt(II), nickel(II) and copper(II) ions to form their corresponding complexes. In all thirty-two metal(II) Schiff bases complexes were isolated. These complexes were characterized by elemental analyses, melting point, IR, EPR, cyclic voltammetry, magnetic susceptibility measurements, differential scanning calorimetry and electronic spectra. The isolation of the unsymmetrical tetradentate Schiff bases and their complexes as well as some of the symmetrical tetradentate Schiff bases and their complexes are considered to be novel. The purity and composition of both the Schiff bases and the metal(II) complexes were established by elemental analyses. The comparison of the IR spectra of the Schiff-bases and their metal complexes indicated that the Schiff bases acted as tetradentate ligands. The observed shifts in the stretching frequencies of (C=N) and (C¯O) are indicative of the formation of these complexes. Further conclusive evidence of the coordination of these Schiff-bases with the metal ions was shown by the appearance of new bands due to (M¯N) and (M¯O) in the metal complexes. Most of the oxovanadium(IV) complexes exhibit a strong band in the range 959−989 cm−1, which have been assigned to (V=O) in a monomeric square pyramidal coordination environment. The i
oxovanadium(IV) complexes with trimethylene bridge, in which their (V=O) appeared at 848−860 cm–1, have been assigned polymeric structure with [V=O∙∙∙·V=O]
interactions,
which
afforded
distorted
octahedral
coordination
geometry. The electronic spectral and magnetic susceptibility measurements were used for assigning the stereochemistry of each metal complex. Electronic spectra indicate a square-planar geometry for all the cobalt(II), nickel(II) and copper(II) complexes. This was also corroborated by the effective magnetic moment of the complexes. The electronic spectra of the oxovanadium(IV) complexes suggest a diversity of geometries. The electronic spectra indicate a square-pyramidal geometry for the fivecoordinate species and distorted octahedral geometry for the six-coordinate species. The room temperature magnetic moments of 1.6–1.8 BM are normal for V(IV) d1 configuration. The solution EPR spectra of the oxovanadium(IV) are consistent with square pyramidal geometry. The cyclic voltammetry of the oxovanadium(IV) complexes revealed only one quasi-reversible wave for each complex and they all showed redox couples with peak- to peak separation values ( Ep) ranging from 74 to 83 mV, indicating a single step one electron transfer process. In vitro glucose uptake was carried out on all the oxovanadium(IV) complexes using C2C12 cell line. All the complexes tested increased glucose utilization in C2C12 cells over basal values except two of the complexes whose percentage glucose uptake was lower than the basal glucose uptake (DMSO). Eighteen of the complexes significantly increased glucose uptake when compared to the basal glucose uptake of the solvent vehicle (DMSO). Cytotoxic test carried out on all the complexes using MTT assay showed that the complexes were not toxic to the cells
ii
at both low and high concentrations. Two of the complexes showed activities comparable or greater than that of insulin. Four unsymmetrical and five symmetrical Schiff base complexes of oxovanadium(IV) have been tested in vivo for their insulin mimetic activities. An acute oral administration of the four unsymmetrical Schiff base complexes of oxovanadium(IV) elicited a progressive reduction in plasma glucose over 6 h in STZ rats. Two of the unsymmetrical Schiff base complexes of oxovanadium(IV) induced a significant reduction in plasma glucose over a 6 h period. Oral administration of the five symmetrical complexes also elicited a progressive reduction in plasma glucose over 6hrs. Two of these complexes induced a significant reduction in plasma glucose during the 6 hour period.
iii
ACKNOWLEDGMENTS I am very much grateful to the Almighty God, the King of kings and the Lord of lords for enabling me to complete this work. I wish to acknowledge my sincere thanks to my supervisors, Prof Gabriel Kolawole and Prof Andy Opoku for their invaluable help, thought-provoking guidance, encouraging attitude and pleasant behaviour throughout this study. I wish to thank the National Research Foundation (NRF), South Africa for their generous financial support of this project. The insulin-mimetic studies would also not have been possible without the help of Dr. Christo Muller of the Diabetic Discovery Platform (DDP), South African Medical Research Council. My sincere thanks also go to the other members of the group (DDP). I also wish to thank Dr. Joanna Wolowska of School of Chemistry, The University of Manchester, UK for running the EPR and Dr. Paresh of Jackson state University, USA for use of DSC instrument. I also wish to thank other lecturers in the department, especially Prof N. Revaprasadu and Mr. G Peckham, for their kind and cooperative behaviour throughout the course of this work. I would like to thank my colleagues in the department, past and present. Special thanks go to Mr. O.M. Odeleye, Mr. O.A. Lawal, Mr. M. Chili, Miss. T. Xaba, Mr. N. Sosibo, Mr. N. Mlambo, Mr. P. Mdluli and Miss. M. Mdlolo. I extend a special thanks to the departmental support staff. I also wish thank Pastor Foli and the members of the Redeem Christian Church of God (Empangeni branch) for their support and their prayers for successful completion of this work. I would like to thank Mrs. Kolawole for her encouragements and prayers throughout the course of this work. I would like to thank my mum and my siblings for their moral support and prayers for successful completion of this work. Special thanks to my dear wife for her patience and encouragement during the course of this work and my son Oluwaseyifunmi for his patience.
iv
CERTIFICATION BY SUPERVISORS
We certify that this work was carried out by Mr. A.A. Nejo in the Department of Chemistry, University of Zululand and is approved for submission in fulfilment of the requirements for the award of the degree of Doctor of Philosophy in Chemistry.
........................................................ Promoter G.A. Kolawole, B. Sc. (Hons), M. Ed, Ph.D (Ibadan), CChem, FRSC Senior Professor of Inorganic Chemistry Department of Chemistry, University of Zululand, Kwadlangezwa, South Africa
..................................................... Co-promoter A.R. Opoku, B. Sc.(Hons) (Knust) Ph.D (Machester) Professor of Biochemistry Department of Biochemistry and Microbiology, University of Zululand, Kwadlangezwa, South Africa
v
DEDICATED To my sons, Oluwaseyifunmi and Oluwatomisin; To my wife, who inspired me a lot; To my mum, who sacrificed every personal comfort to see me through my basic education
vi
LIST OF PUBLICATIONS FROM THIS WORK A A. Nejo, G A. Kolawole, A R. Opoku, J Wolowska, P O’ Brien, “Synthesis, Characterization and preliminary Insulin-enhancing studies of symmetrical tetradentate Schiff Base Complexes of Oxovanadium(IV)” Inorganica Chimica Acta 362 (2009) 3993-4001
A A. Nejo, G A. Kolawole, A R. Opoku, C Muller and J Wolowska, “Synthesis, Characterization and Insulin-enhancing studies of Unsymmetrical tetradentate Schiff Base Complexes of Oxovanadium(IV)” Journal of Coordination Chemistry 62:21 (2009) 3411-3424
vii
ORAL COMMUNICATIONS AND POSTER PRESENTATIONS IN CONFERENCES ORAL COMMUNICATIONS 5th International Symposium on Recent Advances in Environmental Health, Jackson, MS, USA Nejo A. A., Kolawole G. A., and Opoku A. R. “Oxovanadium(IV) Complexes of Symmetrical Schiff Bases as possible Insulin enhancers” September 14th – 17th 2008.
39th SACI National Convention, Stellenbosch, SouthAfrica Nejo A. A., Kolawole G. A., Opoku A. R. and Muller C. “Synthesis, Characterization and Insulin-enhancing studies of Unsymmetrical Tetradentate Schiff Base Complexes of Oxovanadium(IV)” November 30th – December 5th 2008.
POSTER PRESENTATIONS 2nd Annual Faculty of Science and Agriculture Research Symposium, Kwadlangezwa, South Africa Nejo A. A., Kolawole G. A. and Opoku A. R “Synthesis, Characterization and Spectroscopic Studies of Symmetrical Tetradentate Schiff Base Oxovanadium(IV) Complexes as Part of Insulin mimetic Studies, November 1st 2007. 38th International Conference of Coordination Chemistry (Jerusalem-Israel) Nejo A. A., Kolawole G. A. and Opoku A. R “Synthesis, Characterization and Insulinenhancing Studies of Symmetrical Tetradentate Schiff Base Complexes of Oxovanadium(IV), July 20th – July 25th 2008.
viii
TABLE OF CONTENTS PAGE Abstract
i
Acknowledgements
iv
Certification
v
List of Publications from this work
vii
Oral Communications and Poster Presentations in Conferences
viii
Table of contents
ix
List of Figures
xvi
List of Tables
xix
List of Abbreviations
xxi
CHAPTER 1 INTRODUCTION 1.1
Schiff bases
1
1.2
Biological Importance of Schiff Bases
3
1.3
Schiff base metal complexes
4
1.4
History and occurrence of vanadium
15
1.4.1 The chemistry of vanadium
17
1.5
19
Oxovanadium(IV) insulin enhancing agents
1.5.1 Pyronates and pyridinonates
20
1.5.2 Acetylacetonates
22
1.5.3 Picolinates
24
1.5.4 Oxovanadium(IV) salen-type derivatives
25
1.5.5 Dicarboxylate ester–oxovanadium(IV) complexes
26
1.6
27
Other metals used as insulin enhancing agents
ix
1.6.1 Zinc compounds
27
1.6.2 Magnesium compounds
28
1.6.3 Chromium compounds
29
1.7
Toxicity of vanadium compounds
30
1.8
Accumulation of vanadium compounds in the body
30
1.9
Mechanism of action of insulin
31
1.10
Types of diabetes
33
1.11
Prevalence of diabetes in the World
34
1.12
Prevalence of diabetes in South Africa
35
1.13
Scope of this work
37
1.14
Aims and Objectives
37
1.15
Compounds covered in the project
38
CHAPTER 2 PHYSICAL TECHNIQUES USED FOR CHARACTERIZATION
2.1
Electron paramagnetic resonance (EPR)
42
2.1.1 The theory of EPR spectroscopy
43
2.1.2 The Effects of Point Symmetry
46
2.2
47
Magnetic susceptibility
2.2.1 The theory of magnetic susceptibility
48
2.2.2 Types of magnetic behaviour
49
2.2.3 Magnetic properties of transition metal complexes
50
2.3
51
UV-Visible spectrophotometry
2.3.1The Molecular Orbital Approach to bonding in oxovanadium(IV) ion
52
2.4
56
Thermal Analysis (TA)
2.4.1 Types of TA Instruments
57
x
2.5
Cyclic Voltammetry
60
CHAPTER 3 EXPERIMENTAL 3.1 Materials
62
3.2 Materials for insulin-mimetic test
62
3.3 Synthesis
62
3.3.1. Preparation of unsymmetrical Schiff bases
63
3.3.2 Preparation of symmetrical Schiff bases
63
3.3.3 Preparation of oxovanadium(IV) complexes
63
3.3.4. Preparation of the cobalt(II), nickel(II) and copper(II) Complexes
64
3.4 Characterization of the ligands and complexes
64
3.4.1 Microanalysis
65
3.4.2 Melting/decomposition points
65
3.4.3 1H NMR spectra
65
3.4.4 Infrared spectroscopy
65
3.4.5 Electronic absorption spectra
65
3.4.6 Electron paramagnetic resonance (EPR)
66
3.4.7 Magnetic moments
66
3.4.8 Cyclic voltammetry
66
3.4.9. Differential scanning calorimetry (DSC)
66
3.5 Insulin-mimetic activity
67
3.5.1 In vitro studies
67
3.5.1.1 Cell culture
67
3.5.1.2 Viable cell counts
68
3.5.1.3 Glucose uptake determination
68
xi
3.5.1.4 Statistical analysis
69
3.5.2 In vivo studies
69
3.5.2.1 Animals
69
3.5.2.2 Experimental groups
70
3.5.2.3 Collecting of blood samples
70
3.5.2.4. Analysis of data
71
3.5.2.5 Gavage procedure
71
3.5.2.6 Statistical Analysis
71
CHAPTER 4 RESULTS AND DISCUSSION 4.1 Synthesis
72
4.1.1 Unsymmetrical Schiff bases
72
4.1.2 Unsymmetrical Schiff base metal(II) complexes
73
4.1.2.1 Unsymmetrical Schiff base complexes of cobalt(II)
74
4.1.2.2 Unsymmetrical Schiff base complexes of nickel(II)
74
4.1.2.3 Unsymmetrical Schiff base complexes of copper(II)
74
4.1.2.4 Unsymmetrical Schiff base complexes of oxovanadium(IV)
74
4.1.3 Symmetrical Schiff bases and complexes of oxovanadium(IV)
75
4.1.3.1 Benzophenoneimine
75
4.1.3.2 Naphthaldiimines
76
4.1.3.3 Chlorosalicylaldiimines
76
4.1.3.4 Methoxysalicylaldiimine
76
4.1.3.5 Ethoxysalicylaldiimine
77
4.2 1H NMR spectra of the Schiff bases
77
4.2.1 Unsymmetrical Schiff bases
78 xii
4.2.2 Symmetrical Schiff bases
78
4.2.2.1 Benzophenoneimine
79
4.2.2.2 Naphthaldiimines
79
4.2.2.3 Chlorosalicylaldiimines
79
4.1.2.4 Methoxysalicylaldimine
80
4.1.2.5 Ethoxysalicylaldiimine
80
4.3 Infrared spectra
81
4.3.1 Unsymmetrical Schiff bases and their complexes
83
4.3.1.1 Unsymmetrical Schiff base complexes of cobalt(II)
83
4.3.1.2 Unsymmetrical Schiff base complexes of nickel(II)
83
4.3.1.3 Unsymmetrical Schiff base complexes of copper(II)
84
4.3.1.4 Unsymmetrical Schiff base complexes of oxovanadium(IV)
84
4.3.2 Symmetrical Schiff bases and their complexes
85
4.3.2.1 Benzophenoneimine
85
4.3.2.2 Naphthaldiimines
86
4.3.2.3 Chlorosalicylaldiimines
87
4.3.2.4 Methoxysalicylaldiimines
87
4.3.2.5 Ethoxysalicylaldiimines
88
4.4 Electronic spectra of the metal(II) complexes
89
4.4.1 Unsymmetrical Schiff base metal(II) complexes
91
4.4.1.1 Unsymmetrical Schiff base complexes of cobalt(II)
91
4.4.1.2 Unsymmetrical Schiff base complexes of nickel(II)
91
4.4.1.3 Unsymmetrical Schiff base complexes of copper(II)
92
4.4.1.4 Unsymmetrical Schiff base complexes of oxovanadium(IV)
92
4.4.2 Symmetrical Schiff base complexes of oxovanadium(IV)
93
xiii
4.4.2.1 Benzophenoneimine complexes
93
4.4.2.2 Naphthaldiimines complexes
94
4.4.2.3 Chlorosalicylaldiimines complexes
95
4.4.2.4 Methoxysalicylaldiimines complexes
96
4.4.2.5 Ethoxysalicylaldiimines complexes
97
4.5 Magnetic moment of the metal(II) complexes
98
4.5.1 Unsymmetrical Schiff base complexes of cobalt(II)
98
4.5.2 Unsymmetrical Schiff base complexes of nickel(II)
98
4.5.3 Unsymmetrical Schiff base complexes of copper(II)
99
4.5.4 Symmetrical and unsymmetrical Schiff base complexes of oxovanadium(IV)
99
4.6 EPR spectra of oxovanadium(IV) complexes
99
4.6.1 Unsymmetrical Schiff base complexes of oxovanadium(IV)
101
4.6.2 Symmetrical Schiff base complexes of oxovanadium(IV)
101
4.7 Cyclic voltammetry
103
4.8 Thermal analysis of oxovanadium(IV) complexes
103
4.8.1 Unsymmetrical Schiff base complexes of oxovanadium(IV)
104
4.8.2 Symmetrical Schiff base complexes of oxovanadium(IV)
105
4.8.2.1 Benzophenoneimine complexes
105
4.8.2.2 Naphthaldiimines complexes
105
4.8.2.3 Chlorosalicylaldiimines complexes
105
4.8.2.4 Methoxysalicylaldiimines complexes
106
4.8.2.5 Ethoxysalicylaldiimines complexes
107
xiv
CHAPTER 5 INSULIN-MIMETIC STUDIES ON THE OXOVANADIUM(IV) COMPLEXES 5.1 In vitro analysis
108
5.1.1 Unsymmetrical Schiff base complexes of oxovanadium(IV)
109
5.1.2 Symmetrical Schiff base complexes of oxovanadium(IV)
110
5.1.2.1 Benzophenoneimine complexes
110
5.1.2.2 Naphthaldiimines complexes
110
5.1.2.3 Chlorosalicylaldiimines complexes
111
5.1.2.4 Methoxysalicylaldiimines complexes
111
5.1.2.5 Ethoxysalicylaldiimines complexes
111
5.2 In vivo analysis
112
5.2.1 Unsymmetrical Schiff base complexes of oxovanadium(IV)
113
5.2.2 Symmetrical Schiff base complexes of oxovanadium(IV)
114
CHAPTER 6 CONCLUSIONS AND FUTURE PROSPECTS 6.1 Conclusions
116
6.2 Suggestion for future work
119
REFFERENCES
121
APPENDIX I Tables of experimental results
135
APPENDIX II Figures of spectroscopic spectra
153
Bar chart for in vitro analysis
186
Bar chart for in vivo analysis
188
xv
LIST OF FIGURES PAGE Fig. 1.1 Some classes of Schiff base ligands
2
Fig. 1.2 Insulin-mimetic behaviour of vanadium compounds
32
Fig. 1.3 Worldwide map showing the prevalence of diabetes
35
Fig. 2.1 Energy levels for an unpaired electron in a magnetic field
45
Fig. 2.2 Energy level ordering of Ballhausen and Gray
55
Fig. 2.3 Changes in the ordering of the d orbitals in oxovanadium(IV) complexes
56
Fig. 4.1 Preparation of the unsymmetrical Schiff bases and their metal(II) complexes
73
Fig. 4.3.1 IR spectra of complex CoL1 and its ligand H2(naph-Clsal)opd: representative spectra for the unsymmetrical complexes of cobalt(II) and the ligands 153 Fig. 4.3.2 IR spectra of complex NiL4 and its ligand H2(naph-Clsal)opd: representative spectra for the unsymmetrical complexes of nickel(II) and the ligands
153
Fig. 4.3.3 IR spectra of complex CuL1 and its ligand H2(naph-Clsal)opd: representative spectra for the unsymmetrical complexes of copper(II) and the ligands 154 Fig. 4.3.4 IR spectra of complex VOL1 and its ligand H2(naph-Clsal)opd: representative spectra for the unsymmetrical complexes of oxovanadium(IV) and the ligands
154
Fig. 4.3.5 IR spectra of complex VOL5 and its ligand H2bp2en: representative spectra for the benzophenoneimine
155
Fig. 4.3.6 IR spectra of complex VOL8 and its ligand H2naph2en: representative spectra for the naphthaldiimine
155
Fig. 4.3.7 IR spectra of complex VOL13 and its ligand H2Clsal2tn: representative spectra for the chlorosalicylaldiimine
156
xvi
Fig. 4.3.8 IR spectra of complex VOL15 and its ligand H2Omesal2en: representative spectra for the methoxysalicylaldiimine
156
Fig. 4.3.9 IR spectra of complex VOL18 and its ligand H2Oetsal2en: representative spectra for the ethoxysalicylaldiimine
157
Fig. 4.4.1 Electronic spectra of the unsymmetrical cobalt(II) complexes
158
Fig. 4.4.2 Electronic spectra of the unsymmetrical nickel(II) complexes
159
Fig. 4.4.3 Electronic spectra of the unsymmetrical copper(II) complexes
160
Fig. 4.4.4 – 4.4.5 Electronic spectra of the unsymmetrical oxovanadium(IV) complexes 161–162 Fig. 4.4.6 – 4.4.7 Electronic spectra of the benzophenoneiminato oxovanadium(IV) complexes
163–164
Fig. 4.4.8 – 4.4.8 Electronic spectra of the napthaldiiminato oxovanadium(IV) complexes 165–166 Fig. 4.4.9 – 4.4.10 Electronic spectra of the chlorosalicylaldiiminato oxovanadium(IV) complexes 167–168 Fig. 4.4.11– 4.4.12 Electronic spectra of the methoxysalicylaldiiminato oxovanadium(IV) complexes
169–170
Fig. 4.4.13 – 4.4.14 Electronic spectra of the ethoxysalicylaldiiminato oxovanadium(IV) Complexes
171–172
Fig. 4.5.1 EPR spectra of oxovanadium(IV) complexes 1[VOL1], 2[VOL2], 3[VOL3], and 4[VOL4]
173
Fig. 4.5.2 EPR spectra of oxovanadium(IV) complexes 5[VOL5], 6[VOL6], 7[VOL7] and 8[VOL12]
174
Fig. 4.6 Cyclic voltammograph of the oxovanadium(IV) complexes
175
Fig. 4.7.1.1– 4.7.5.3 DSC curves of oxovanadium(IV) complexes VOL1 – VOL20
176–186
Fig. 5.1.1 Glucose uptake graph for the oxovanadium(IV) complexes VOL1 – VOL10
186
Fig. 5.1.2 Glucose uptake graph for the oxovanadium(IV) complexes VOL11 – VOL20
187
xvii
Fig. 5.2.1 The effect of the unsymmetrical complexes on hyperglycemia in Wistar outbred rats with STZ-induced diabetes
188
Fig. 5.2.2 The effect of five symmetrical complexes on hyperglycemia in Wistar outbred rats with STZ-induced diabetes
189
xviii
LIST OF TABLES PAGE Table 1.1 List of countries with the highest numbers of estimated cases of diabetes for 2000 and 2030
36
Table 1.2 Prevalence estimates of diabetes in South Africa: comparison of years 2003 and 2025
36
Table 1.3 Nomenclature and formulae for the Schiff base ligands
39
Table 1.4 Nomenclature and formulae for the oxovanadium(IV) complexes
40
Table 2.1 Relationships between g and A tensors, EPR symmetry and the point symmetry of paramagnets
46
Table 2.2 The expected spin only moments for n unpaired electron system
51
Table 4.1.1 Physical properties and analytical data for the Schiff bases and oxovanadium(IV) complexes
135
Table 4.1.2 Physical properties and analytical data for cobalt(II), nickel(II) and copper(II)the compounds
137
Table 4.2 1H NMR data for free ligands
138
Table 4.3.1 Selected infrared spectral bands of the Schiff bases and oxovanadium(IV) complexes
139
Table 4.3.2 Selected infrared spectral bands of cobalt(II), nickel(II) and copper(II) complexes
141
Table 4.4.1 Electronic spectral data of the cobalt(II) complexes
142
Table 4.4.2 Electronic spectral data of the nickel(II) complexes
143
Table 4.4.3 Electronic spectral data of the copper(II) complexes
144
Table 4.4.4 Electronic spectral data of the oxovanaduim(IV) complexes
145
Table 4.5 Room temperature magnetic moments for the complexes
148
Table 4.6.1 EPR parameters for the oxovanadium(IV) complexes with axial symmetry
149
Table 4.6.2 EPR parameters for the oxovanadium(IV) complexes with xix
rhombic symmetry
149
Table 4.7 Cyclic voltammetric data for oxovanadium(IV) complexes
150
Table 4.8 DSC phenomenological data of the complexes
151
Table 5 Glucose uptake data for the oxovanadium(IV) complexes
152
xx
LIST OF ABBREVIATIONS AMPK
AMP-activated protein kinase
ATCC
American Type Culture Collection Bohr magneton
BM
Bohr magneton
BEOV
Bis(ethylmaltolato)oxovanadium(IV)
BMOV
Bis(maltolato)oxovanadium(IV)
CoII(salen)
N,N’-bis(salicylaldene)ethylenediaminatocobalt(II)
CoII(salpn)
N,N’-bis(salicylaldene)propylenediaminatocobalt(II)
o
degree Celsius
CN−
cyano
CV
Cyclic Voltammetry
DMF
dimethylformamide
DMSO
dimethylsulphoxide
DSC
differential scanning calorimetry
DTA
differential thermal analysis
EPR
electron paramagnetic resonance
FBS
fetal bovine serum
FFA
free fatty acid
GLUT
Glucose transporters
g
gyromagnetic ratio
h
Planck’s constant
H
magnetic field
Hema
ethylmaltol, 3-hydroxy-2-ethyl-4-pyrone
Hdpp
1,2-dimethy-3-hydroxy-4-pyridinone
C
xxi
Hkoj
kojic acid, 5-hydroxy-2-hydroxymethy-2-pyrone
Hma
maltol, 3-hydroxy-2-methyl-4-pyrone
IDDM
insulin-dependent diabetes mellitus
IDF
International Diabetes Federation
IR
Infrared
LMCT
ligand to metal charge transfer
MeOH
methanol
M
molarity or metal ion
MIC
minimum inhibitory concentrations
NIDDM
non-insulin-dependent diabetes mellitus
NMR
nuclear magnetic resonance
nm
nanometre
NiII(salen)
N,N’-bis(salicylaldene)ethylenediiminatonickel(II)
NiII(salpn)
N,N’-bis(salicylaldene)propylenediiminatonickel(II)
PTK
protein–tyrosine kinase
PTP
protein tyrosine phosphatase
py
pyridine
STZ
streptozotocin
TGA
Thermogravimetric Analysis
TNAPY
N-(2-thienylmethylidene)-2-aminopyridine
ìe
effective magnetic moment
UV
ultraviolet
VO(acac)2
bis(acetylacetonato)oxovanadium(IV)
VO(ma)2
bis(maltolato)oxovanadium(IV)
VOMPA
bis(6-methylpyridine-2-carboxylato)oxovanadium(IV)
xxii
VO(pic)2
bis(picolinato)oxovanadium(IV)
Vis
visible
Zn(6mpa)2
zinc(II)–6-methylpicolinate
Zn(pa)2
zinc(II)–picolinate
Zn(6mpa-ma)2
bis(6-methylpicolinemethylamido)zinc(II)
xxiii
CHAPTER 1 INTRODUCTION 1.1 Schiff bases Schiff bases are typically formed by the condensation of a primary amine and an aldehyde/ketone. The resultant compound, R1R2C=NR3, is called a Schiff base (named after Hugo Schiff), where R1 is an aryl group, R2 is a hydrogen atom and R3 is either an alkyl or aryl group. However, usually compounds where R3 is an alkyl or aryl group and R2 is an alkyl or aromatic group are also regarded as Schiff bases. Schiff bases that contain aryl substituents are substantially more stable and more readily synthesized, while those which contain alkyl substituents are relatively unstable. Schiff bases of aliphatic aldehydes are relatively unstable and readily polymerizable [1], while those of aromatic aldehydes having effective conjugation are more stable. In general, aldehydes react faster than ketones in condensation reactions, leading to the formation of Schiff bases as the reaction centre of aldehyde are sterically less hindered than that of ketone. Furthermore, the extra carbon of ketone donates electron density to the azomethine carbon and thus makes the ketone less electrophilic compared to aldehyde [2]. Schiff bases are generally bidentate (1), tridentate (2), tetradentate (3) or polydentate (4) ligands capable of forming very stable complexes with transition metals. They can only act as coordinating ligands if they bear a functional group, usually the hydroxyl, sufficiently near the site of condensation in such a way that a five or six membered ring can be formed when reacting with a metal ion (Fig. 1.1). The tetradentate Schiff base class is of the type reported in this thesis.
1
Schiff bases derived from aromatic amines and aromatic aldehydes have a wide variety of applications in many fields, e.g., biological, inorganic and analytical chemistry [3, 4]. Applications of many new analytical devices require the presence of organic reagents as essential compounds of the measuring system.
OH R C
OH R
C N
R
N NR1 2
1
Bidentate (1 ) Tridentate (2 ) CH3 OH
R
C N
HO N C
R
N
R
C
C
OH
OH
R
N HO
Pentadentate (4)
Tetradentate (3)
Fig. 1.1 Some classes of Schiff base ligands
Schiff bases are used, e.g., in optical and electrochemical sensors, as well as in various chromatographic methods, to enable detection of enhanced selectivity and sensitivity [5-7]. Among the organic reagents actually used, Schiff bases possess excellent characteristics, structural similarities with natural biological substances, relatively simple preparation procedures and the synthetic flexibility that enables design of suitable structural properties [8, 9]. Schiff bases are widely applicable in 2
analytical determination, using reactions of condensation of primary amines and carbonyl compounds in which the azomethine bond is formed (determination of compounds with an amino or carbonyl group); using complex formation reactions (determination of amines, carbonyl compounds and metal ions); or utilizing the variation in their spectroscopic characteristics following changes in pH and solvent [10]. Schiff bases play important roles in coordination chemistry as they easily form stable complexes with most transition metal ions [11, 12]. In organic synthesis, Schiff base reactions are useful in making carbon-nitrogen bonds.
1.2 Biological Importance of Schiff Bases Schiff bases appear to be important intermediates in a number of enzymatic reactions involving interaction of the amino group of an enzyme, usually that of a lysine residue, with a carbonyl group of the substrate [13]. Stereochemical investigations [14] carried out with the aid of molecular models showed that Schiff bases formed between methylglyoxal and the amino group of the lysine side chains of proteins can bend back in such a way towards the N atom of peptide groups that a charge transfer can occur between these groups and the oxygen atoms of the Schiff bases. Schiff bases derived from pyridoxal (the active form of vitamin B6) and amino acids are considered as very important ligands from biological point of view. Schiff bases are involved as intermediates in the processes of non-enzymatic glycosylations. These processes are normal during aging but they are remarkably accelerated in pathogeneses caused by stress, excess of metal ions or diseases such as diabetes, Alzheimer’s disease, and atherosclerosis. Non-enzymatic glycosylation begins with an attack of sugar carbonyls or lipid peroxydation fragments on amino groups of proteins, aminophospholipids and nucleic acid, 3
causing tissue damages by numerous oxidative rearrangements. One of the consequences is cataract of lens proteins [15]. Many biologically important Schiff bases have been reported in the literature possessing, antimicrobial, antibacterial, antifungal, anti-inflammatory, anticonvulsant, antitumor and anti HIV activities [16-21]. Another important role of Schiff base structure is in transamination [22]. Transamination reactions are catalyzed by a class of enzymes called transaminases. Transaminases are found in mitochondria and cytosal of eukaryotic cells. All the transaminases appear to have the same prosthetic group, i.e., pyridoxal phosphate, which is covalently attached to them via an imino group. Schiff base formation is also involved in the chemistry of vision, where the reaction occurs between the aldehyde function of 11-cis-retinal and amino group of the protein (opsin) [23].
The biosynthesis of porphyrin, for which glycine is a
precursor, is another important pathway, which involves the intermediate formation of Schiff base between keto group of one molecule of δ-aminolevulinic acid and εamino group of lysine residue of an enzyme.
1.3 Schiff base metal complexes Transition metals are known to form Schiff base complexes and Schiff bases have often been used as chelating ligands in the field of coordination chemistry. Their metal complexes have been of great interest for many years. It is well known that N and S atoms play a key role in the coordination of metals at the active sites of numerous metallobiomolecules [24]. Schiff base metal complexes have been widely studied because they have industrial, antifungal, antibacterial, anticancer, antiviral and herbicidal applications [25-30]. They serve as models for biologically important species and find applications in biomimetic catalytic reactions. Chelating ligands containing N, S and O donor atoms show broad biological activity and are of special 4
interest because of the variety of ways in which they are bonded to metal ions. It is known that the existence of metal ions bonded to biologically active compounds may enhance their activities [28-30]. Schiff base metal complexes have been known since the mid nineteenth century [31] and even before the general preparation of the Schiff bases ligands themselves. Schiff base metal complexes have occupied a central place in the development of coordination chemistry after the work of Jørgensen and Werner [32]. However, there was no comprehensive, systematic study until the preparative work of Pfeiffer and associates [33]. Pfeiffer and his co-workers [34] reported a series of complexes derived from Schiff bases of salicylaldehyde and its substituted analogues. Structure and mechanism of the formation of the Schiff base complexes and stereochemistry of four coordinate chelate complexes formed from Schiff bases and their analogues have been discussed in several reviews [35]. The configuration of the chelate group in the four coordinate complexes may be square-planar, tetrahedral, distorted tetrahedral or distorted trigonal pyramidal with the metal atom at the apex. The configuration depends primarily on the nature of the metal atom and also on the magnitude and symmetry of the ligand field. Metal complexes have also been reported with other ligands mixed with Schiff bases. Of all the Schiff base complexes, those derived from salicylaldiimines have been thoroughly studied so far. A variety of physiochemical investigations on these complexes provide a clear understanding of their stereochemical and electronic properties. The advantage of the salicylaldiimines ligand systems is the considerable flexibility of the synthetic procedures, which have resulted in the preparations of a wide variety of complexes with a given metal whose properties are often dependent on the ligand structure.
5
This review will concentrate on metal Schiff bases of some of the first row transition metals with emphasis on tetradentate Schiff base complexes of oxovanadium(IV), cobalt, nickel and copper A number of structural studies on the effect of the number of CH2 groups between the two azomethine moieties in VO2+, Co2+, Ni2+, Cu2+ and Zn2+ complexes of tetradentate Schiff bases (5) derived from salicylaldehyde and a variety of diamines (1:2 ratio) have been reported [36-39]. They have been shown that an increase in the methylene chain length allows adequate flexibility for the complexes to change their structure from planar towards a distorted or pseudo-tetrahedral coordination depending on the magnitude of n. In addition, the longer chains cause the ligand field strength to decrease [36, 37]. Metal complexes of this type have been prepared for the series n = 2 to 10 for the bivalent cobalt, nickel, copper, zinc and manganese. For n = 2 most divalent first-row transition metals are expected to form square-planar complexes. However, a decrease in ligand field strength has been reported for nickel(II), copper(II) and zinc(II) derivatives as n is increased from 2 to 4. This decrease corresponds to an increase in distortion from planarity with increase in the length of the methylene bridge. Kolawole and Patel [40] synthesized a series of [VO{OC6H4CH=NCR1R2(CH2)n-1-N=CHC6H4O}] complexes, where n = 2–10. The ν(V=O) stretching frequencies fall in the range 861–994 cm−l and the effective magnetic moments at room temperature of the complexes are between 1.64 and 1.81 BM. The complexes with [(n = 2; R1 = R2 = H), (R1 = H, R2 = CH3), (R1 = R2 = CH3)] are green, and their spectroscopic and magnetic properties suggest that they have tetragonal pyramidal structures. A corresponding complex (R1 = R2 = H, n = 3) is orange-yellow and its Xray structure shows that it is polymeric, having a distorted octahedral geometry. The 6
electronic spectra in chloroform and pyridine and in the solid state indicate the possibility of an inversion of the ordering of energies of the eπ* and b1* levels; consequently a diversity of geometries were observed as the methylene chain length increased. The stereochemistry of the complexes varies from distorted squarepyramidal to octahedral geometry. In continuation of their study on oxovanadium(IV) ion, Kolawole, et al [41-43] also
synthesized
oxovanadium(IV)
complexes
of
tetradentate
3-
methoxysalicylaldiimine, 5-chlorosalicylaldiimine and naphthaldiimine Schiff bases containing the N2O2 chromophore and long-chain alkyl equatorial bridges. Their spectroscopic and magnetic properties suggest that the complexes are fivecoordinate, except the trimethylene derivatives (having a six-membered equatorial ring size above V) are orange-yellow and polymeric. Substitution in the aromatic rings did not affect significantly the trend in the stereochemistry of the complexes. The 3-methoxysalicylaldiimine complexes of oxovanadium(IV) reported by Patel and Kolawole [41] is an isomeric form of 5-methoxysalicylaldiimine complexes reported in this thesis.
O
O MII N HC
CH N (CH2 )n 5
In general, Co(II) complexes have a higher tendency to assume a tetrahedral configuration than the corresponding Ni(II) complexes. The CoII(salen) complex has a low spin square planar structure with a single unpaired electron and a magnetic moment of about 2.5 BM [39], and it is extremely oxygen sensitive. Increasing the 7
number of the methylene units in diamine chain of the Schiff base ligand (5) allows the Co(II) complexes to change from a square planar (n = 2) to a tetrahedral geometry [37, 38]. While the CoII(salen) complex has no geometrical distortion with respect to the primary ligand, CoII(salpn) has a distorted structure. This is in contrast to the Ni(II) series, which maintains a square planar geometry irrespective of increase in the number of the methylene groups, or adopts an octahedral geometry in the presence of donor solvents like MeOH, DMF, DMSO [36, 37]. The distortion about the cobalt centre is due to the extra methylene groups which causes an apparent weakening of the ligand field strength. In other words, the increasing chain length in salpn allows more flexibility to form a tetrahedral coordination geometry about the cobalt [the cobalt(II) shows a more pronounced tendency for the formation of tetrahedral complexes than either Ni(II) or Cu(II)] [44]. Therefore, CoII(salpn) complex shows a low tendency to increase its coordination number by forming octahedral complexes in the presence of addition donors [44, 45]. However, relatively strong π-acceptor ligands (for example pyridine and its derivatives, or anions such as N3−, CN−) increase effectively the ligand field strength. Bis(N-alkyl- or bis(N-aryl-salicylideneiminato)nickel(II) complexes are basically 4-coordinate square-planar in the solid state. In chloroform or benzene Ni(II) salicylideneiminato-complexes become partially paramagnetic [46]. This effect arises through the monomeric planar speices being in equilibrium with dimeric or polymeric or tetrahedral form, which are paramagnetic [46]. Paramagnetic form of the complex of N-methylsalicyladiimine with Ni(II) has been isolated in solid state confirming that polymeric species are involved. As the length n increases from 2, the ligand field produced becomes much weaker, as indicated by the shift of the main d–d band towards lower wavenumbers and by lowering of the capacity of Ni(II) to bind
8
additional ligands, such as py, at the fifth and sixth coordination sites [46]. For instance, when dissolved in pyridine, [NiII(salen)] maintains 4-coordinate planar configuration binding no additional pyridine molecules, while [NiII(saltn)] binds two pyridine molecules to form 6-coordinate bis(pyridine)nickel(II) complex. Few studies have been reported where Ni(II) complexes are diamagnetic and planar. The Ni(II) complexes of N-isopropylsalicyladiimines and of its 5-ethyl derivative are tetrahedral and paramagnetic, whereas the complexes of 5-methyl-, 5-n-propyl-, 5-chloro- and 5-nitro-N-isopropyl salicyaldiimine are all planar and diamagnetic [46]. The presence of long side chains on the ligands tends to lower the symmetry of the nickel complexes, which in turn changes the stereochemistry of the complexes. The complexity of stereochemistry of copper(II) complexes has been well documented, and many factors, such as ligand field stabilization energies, the Pauling electroneutrality principle, the Jahn-Teller effect, steric effects, etc., have been invoked to account for the complicated structures [47]. Copper(II) complexes of (5) suffer greater increasing distortion from planarity towards tetrahedral geometry as n
becomes
large.
X-ray
diffraction
studies
have
revealed
that
[N,
N’-
bis(salicylidene)ethylenediiminatocopper(II)] is dimeric, resulting in some out-ofplane distortion due to a weak intermolecular Cu—O bond [39]. This makes each copper atom five-coordinate with a near square-pyramidal arrangement of ligand donor atoms. Many complexes of the Cu(sal·R)2 type, where R denotes alkyl or aryl group have been reported and the complexes are considered to be square-planar [39]. Some of them, however, consist of binuclear units, in which there is very weak copper to copper bonds [39]. Cu-(Sal·R)2, where R is alkyl or aryl groups, has square-planar configuration and is stable, but when there is considerable steric
9
hindrance, the distortion from the planarity may occur. Thus for R = tert-C4H9, isoC3H7 and cyclohexyl, it was concluded from the electronic spectra that the complexes are distorted from the square plane [39]. It was also concluded that the distortion decreases in the following order tert-C4H9 > iso-C3H7 > C6H11. This was confirmed by X-ray studies, which showed that the angles between the two N-Cu-O planes is 80o for the complex with R = tert-C4H9 and 60o for the complex with R = iso-C3H7. Schiff base of 4-aminoantipyrine and its complexes have a variety of applications in biological, clinical, analytical and pharmacological areas. Raman, et al. [48] prepared a new series of transition metal complexes of Cu(II), Ni(II), Co(II), Mn(II), Zn(II), VO(IV), Hg(II) and Cd(II) from the Schiff base derived from 4aminoantipyrine, 3-hydroxy-4-nitrobenzaldehyde and o-phenylenediamine. Structural features were obtained from their elemental analyses, magnetic susceptibility, molar conductance, IR, UV–Vis, 1H NMR and EPR spectral studies. The data show that these complexes have composition of ML type. The UV–Vis, magnetic susceptibility and EPR spectral data of the complexes suggest square–planar geometry around the central metal ion except VO(IV) complex which has square–pyramidal geometry. The redox behaviour of the copper and oxovanadium(IV) complexes was studied by cyclic voltammetry. Antimicrobial screening tests gave good results for the metal complexes. The nuclease activity of the above metal complexes shows that Cu, Ni and Co complexes cleave DNA through redox chemistry whereas other complexes were not effective. The complexes of Cr(III), Fe(III), Co(II) and Ni(II) ions with a Schiff base derived from 4–dimethylaminobenzaldehyde and primary amines have been prepared and investigated using different physico-chemical techniques, such as
10
elemental analysis, molar conductance measurements, and infrared and electronic spectra. The chemical analysis data showed the formation of the complexes and a square planar geometry was suggested for Co(II) and Ni(II) complexes and an octahedral structure for Cr(III) and Fe(III) complexes [49]. Nair,
et
al.
[50]
synthesized
two
Schiff
bases
from
5-ethyl-2,4-
dihydroxyacetophenone. Their copper, nickel, iron and zinc complexes were screened for antibacterial activity against some clinically important bacteria, such as Pseudomonas
aeruginosa,
Proteus
vulgaris,
Proteus
mirabilis,
Klebsiella
pneumoniae and Staphylococcus aureus. The Schiff bases showed greater activity than their metal complexes. The metal complexes showed differential effects on the bacterial strains investigated and the solvent used, suggesting that the antibacterial activity is dependent on the molecular structure of the compound, the solvent used and the bacterial strain under consideration.
Amongst the four metals, Zn
complexes showed the best antibacterial activity followed by Fe in 1,4-dioxane while Ni followed by Zn and Fe showed the best antibacterial activity in DMF. Metal complexes, ML2Cl2, where M is Fe(II), Co(II), Ni(II), Cu(II), Zn(II), or Cd(II),
and
L
is
the
thiophenecarboxaldehyde
Schiff with
base
formed
2-aminopyridine,
by
condensation
of
2-
N-(2-thienylmethylidene)-2-
aminopyridine (TNAPY), have been prepared and characterized by elemental analysis and magnetic and spectroscopic measurements [11]. Elemental analysis of the chelates suggests the stoichiometry is 1:2 (metal-ligand). Infrared spectra of the complexes agree with the coordination to the central metal atom through the nitrogen of the azomethine (—HC=N—) group and the sulfur atom of the thiophene ring. Magnetic susceptibility data coupled with electronic and EPR spectra suggest a distorted octahedral structure for the Fe(II), Co(II), Ni(II), and Cu(II) complexes, and
11
a tetrahedral geometry for the Zn(II) and Cd (II) complexes. The Schiff base and its metal chelates were also screened for their in vitro antibacterial activity against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. The metal chelates were shown to possess more antibacterial activity than the uncomplexed Schiff-base. Raman, et al. [51] have synthesized new Schiff base chelates of Cu(II), Co(II), Ni(II) and Zn(II) derived from benzil-2,4-dinitrophenylhydrazone with aniline. EPR spectral studies were carried out to suggest tentative structures for the complexes. The electronic absorption spectra of the Schiff base and its Cu(II), Co(II), Ni(II) and Zn(II) complexes were recorded at room temperature using acetone as solvent. The electronic spectra and magnetic susceptibility data of the complexes suggest octahedral geometry around the central metals ion. Ni(II), Cu(II), Pd(II) and Pt(II) complexes of thiophene-2-carboxaldehyde Schiff bases of S-methyl- and S-benzyl dithiocarbazate have also been reported [52]. Magnetic and spectroscopic evidences support a square-planar structure for these complexes. The crystal structure of nickel and copper were determined by X−ray diffraction. Both complexes have a trans-planar structure in which the two Schiff base ligands are coordinated to the metal(II) ions as uninegatively charged bidentate ligands via the thiolate S and the azomethine N atoms. These complexes were also screened for their antifungal activity. Ni(II) Schiff base complex derived from salicylaldehyde and o-aminobenzoic acid has been prepared and characterized [53]. The elemental analysis data show the formation of 1:1 [M:L] complex. The molar conductance measurement reveals a non-electrolytic nature. The electronic spectrum of the complex was typical of a square planar geometry. It was diamagnetic. The free Schiff base and its complex
12
were tested for antibacterial activities against several human pathogenic bacteria. They show no detectable activity against any of the bacteria screened [53]. Complexes of Ni(II), Co(II) and Cu(II) with Schiff base ligands derived from βdiketones and p-anisidine have been reported [54]. Spectral and magnetic studies on these complexes indicate that they are four coordinate, with square-planar geometry. It has been found that all the complexes are antimicrobially active and show higher activity than the free ligands. Recently, there has been increasing interest in the synthesis and characterization of unsymmetrical Schiff base ligands and their metal complexes. This is due partly to the belief that the systematic investigation of these complexes may shed light on the nature of complexes of biological interest [55]. Unsymmetrical tetradentate Schiff base complexes are required to model the irregular binding of peptides because trace metals have been found to occur in metalloenzymes bound to a macrocycle such as the heme ring, or to donor atoms of peptide chains usually in a distorted environment [56]. Unsymmetrical ligands can clearly offer many advantages over their symmetrical counterparts in the elucidation of the composition and geometry of metal ion binding sites in metalloproteins and in the development leading to the duplication of enzymatic efficiency and selectivity of natural system with synthetic materials. A large percentage of enzymes have a metal atom at the active site. These metalloenzymes facilitate a variety of reactions, which include redox reaction (carried out by the oxidases and oxygenases), acid-catalysed hydrolysis (hydrolases) and rearrangement of carbon-carbon bonds (synthases and isomerases) [57, 58]. Osowole, et al. [59] prepared Ni(II), Cu(II) and Zn(II) complexes of unsymmetrical Schiff base derived from 2-hydroxy-1-naphthaldehyde, 2, 413
pentanedione and p-phenylenediamine and their adducts with 2,2΄-bipyridine and 1,10-phenanthroline. The magnetic moments and electronic spectra corroborate octahedral geometry for Ni(II) and Cu(II) complexes whereas the Zn(II) Schiff base complex analysed as 4-coordinate and the adduct as 6-coordinate. The antimicrobial properties
of
the
ligand
and
complexes
against
Staphlococcus
aureus,
Streptococcus faecalis, Bacillus sp, Escherichia coli, Pseudomonas aeruginosa, Salmonella tyhi, Klebsiella pneumonia, Acinetobacter sp, Flavobacterium sp, Enterococcus faecalis and Candida albicans were reported.
The compounds
generally exhibited good activity against the selected organisms. The Cu(II) complex has comparable activity to gentamycin. The minimum inhibitory concentrations (MICs) of the sensitive compounds were also reported. Copper(II) complexes of five new tetradentate unsymmetrical ligands, ethylene-N-(acetylacetoneimine)-Nʹ-(ortho-hydroxyarylaldimine) (aryl = 3,5-dibromo phenyl,
3-methylphenyl,
3,6-dimethylphenyl,
3,5-dichlorophenyl,
3,5-dibromo
acetophenone) have been reported [60]. The general coordination configuration was revealed by an X-ray crystallographic study of one of the complexes, ethylene-N(acetylacetoneiminato)-Nʹ-(ortho-hydroxy-3,5-dibromoacetophenoneiminato) copper(II), which exhibits an approximately square-planar geometry with a slight tetrahedron distortion. The substituent effect studied by EPR indicates that electronic factors have a profound effect on the central metal ions. Thermal analysis indicates that both salicylaldiiminato and acetylacetoneiminato donating moieties are thermally compatible. Two series of novel unsymmetrical tetradentate Schiff bases derived from ophenylenediamine
and
1,3-naphthalenediamine
and
their
oxovanadium(IV)
complexes were synthesized by template and non-template methods and
14
characterized by elemental analysis, IR, 1H and
13
C NMR and UV-Vis [61]. These
complexes were used as catalysts for the selective aerobic oxidation of cyclohexene. The catalytic activity increases as the number of electron-donor groups decreases. Complexes containing the naphthylene-bridged ligands had similar redox potential. However, their catalytic activities were quite varied and the differences in their activities were strongly dependent on the fine structural data and redox potential. But in the complexes containing phenylene-bridged ligands, yield a good correlation between catalytic activity and redox potential.
1.4 History and occurrence of vanadium Vanadium has atomic number 23. It is a soft, silvery grey, ductile transition metal. Vanadium was originally discovered by Andrés Manuel del Río, a Mexican mineralogist, in 1801. He first named it panchromium, because of the varied colours of its salts, but changed the name later on to erythronium (‘red’) as a reference to the red colour of its salts when treated with acids [62, 63]. However, soon he withdrew his discovery, since a French chemist incorrectly declared that this new element was only impure chromium. Vanadium was rediscovered in 1831 by the Swedish chemist Nils Gabriel Sefström (1787-1845) in remnants of iron ore quarried at the Taberg in Småland. He named the element vanadin, after the goddess of beauty, youth and love, Vanadis, referring to the beautiful multicoloured compounds [64].
After
Sefström announced the discovery of vanadium, the brown lead ore from Mexico was reanalysed and it was shown that it really contained vanadium instead of chromium. Natural vanadium is a mixture of two isotopes,
51
V (99.76%) and
50
V
(0.24%), the latter being slightly radioactive with a half-life of >3.9 x 1017 years. Today, vanadium is primarily obtained from the minerals vanadinite [Pb5(VO)3Cl] and 15
carnotite [K2(UO2)2(VO4)2] by heating crushed ore in the presence of carbon and chlorine to produce vanadium trichloride. The vanadium trichloride is then heated with magnesium in an argon atmosphere. It is also present in some crude oils in the form of organic complexes. Vanadium is corrosion resistant and is sometimes used to make special tubes and pipes for the chemical industry. Vanadium also does not easily absorb neutrons and has some applications in the nuclear power industry. A thin layer of vanadium is used to bond titanium to steel. Nearly 80% of the vanadium produced is used to make ferrovanadium or as an additive to steel. Ferrovanadium is a strong, shock resistant and corrosion resistant alloy of iron containing between 1% and 6% vanadium. Ferrovanadium and vanadium-steel alloys are used to make such things as axles, crankshafts and gears for cars, parts of jet engines, springs and cutting tools. Vanadium(V) oxide is perhaps vanadium's most useful compound. It is used as a mordant, a material which permanently fixes dyes to fabrics. Vanadium(V) oxide is also used as a catalyst in the contact process and in the manufacture of ceramics. Vanadium(V) oxide can also be mixed with gallium to form superconductive magnets. Vanadium occurs with an abundance of 0.014% in the earth’s crust and is widespread [65]. The element is the second most abundant transition metal in the oceans (50 nM) [65]. Some aquatic organisms are known to accumulate vanadium. For instance, members of an order of tunicates (Ascidiacea) concentrate vanadium to 0.15 M in specialised blood cells [66]. However, the actual function of vanadium and the nature of the vanadium compounds present in these organisms remains unclear [62].
A naturally occurring vanadium-containing enzyme, vanadium
16
bromoperoxidase was discovered in the marine brown alga, Ascophyllum nodosum [67]. Since then, several vanadium haloperoxidases have been isolated and studied [68, 69]. Many of these enzymes have been detected in brown and red seaweeds [70, 71]. However, the accumulation of vanadium is not restricted to marine organisms, since vanadium containing haloperoxidases have also been isolated from terrestrial fungi [72] and a vanadium compound of low molecular weight (amavadin) has been isolated from the toadstool Amanita muscaria [73].
1.4.1 The chemistry of vanadium Vanadium has an electronic configuration of [Ar]3d34s2 and can exist in eight oxidation states ranging from –3 to +5, but with the exception of –2 [74]. Only the three highest, +3, +4 and +5, are important in biological systems [75-77]. Under ordinary conditions, the +4 and +5 oxidation states are the most stable [74]. The coordination chemistry of vanadium is strongly influenced by the oxidation/reduction properties of the metal centre and the chemistry of vanadium ions in aqueous solution is limited to oxidation states of +2, +3, +4 and +5. Vanadium compounds of oxidation state of +2 and +3 are unstable to air and their compounds are predominantly octahedral. Many oxovanadium(V) complexes contain the VO2+ entity and the cis geometry in dioxo complexes have been confirmed by structural determination [78]. The oxo complexes of the halides, alkoxides, peroxides, hydroxamates and amino carboxylates have been characterized [79]. The oxidation of ligands by vanadium(V) prevents the isolation of a larger number of complexes. On the other hand, the oxidizing properties of vanadium(V) compounds are useful for many preparative reactions, namely for the catalysis of oxidations. Important
17
examples are catalyst used for the oxidation of SO2 to SO3 in the industrial production of sulphuric acid Vanadium(IV) is the most stable oxidation state under ordinary conditions and majority of vanadium(IV) compounds contain the VO2+ unit (oxovanadium(IV) or vanadyl ion) which can persist through a variety of reactions and in all physical states. The VO2+ ion forms stable anionic, cationic and neutral complexes with several types of ligands and has one coordination position occupied by the vanadyl oxygen. A wide variety of oxovanaduim(IV) complexes have been prepared and characterized [74, 80]. A square pyramidal geometry has been well established with the oxovanadium(IV) oxygen apical and the vanadium atom lying above the plane defined by the donor atoms of the equatorial ligands. These square pyramidal complexes generally exhibit strong tendency to remain five coordinate [80]. However, orange polynuclear linear chain structures (···V=O···V=O···) [81, 82] and orange octahedral structures with a weak coordination of a solvent molecule are observed in the solid state for the Schiff base-oxovanadium(IV) complexes which have a six-membered N-N chelate ring. These complexes take a distortedoctahedral coordination. The absorption band due to V=O stretching vibration of oxovanadium(IV) complexes is usually observed at a higher wavenumber compared to those of vanadate(V) complexes. The V=O stretching vibration, however, is susceptible to a number of influences including electron donation from basal plane ligand atoms, solid-state effects, and coordination of additional molecules. Therefore, there has been considerable work done to assign the V=O stretching frequencies in oxovanadium(IV) compounds [83-85]. Electronic absorption spectra of oxovanadium(IV) complexes are normally interpreted in terms of the energy level scheme derived from a molecular orbital
18
treatment for a square-pyramidal structure with C4v symmetry at the metal center, [86, 87] in which the z-axis is taken as the vanadium–oxygen double bond, and the x- and y-axes are taken along the equatorial bonds. In this scheme, b2 (dxy) < eπ* (dxz, dyz) < b1* (dx2-y2 ) < a1* (dz2), three electronic transitions are predicted, and indeed three absorption bands due to the d–d transitions are usually observed for oxovanadium(IV) complexes [83, 84, 88]. However, in case of distorted oxovanadium(IV) complex, four absorption bands are observed owing to the splitting of dxz and dyz [88]. The absorption bands that have extinction coefficients in the thousands and which are assigned as ligand to metal charge transfer (LMCT) are sometimes observed. This is elaborated further in Chapter 2. Due to the d1 configuration of V(IV) ions, vanadium(IV) species are easily identified by EPR spectroscopy. Typical eight-line patterns are observed due to hyperfine interaction of the 51V nucleus (I = 7/2) [89].
1.5 Oxovanadium(IV) insulin enhancing agents Before the discovery of insulin and its clinical trials for treating diabetes mellitus (DM), inorganic salts of vanadium have long been known to act as orally viable mimics or enhancing agents for increased activity of insulin in vitro and in vivo. The first report of vanadium salts being used as a metallotherapeutic agent appeared in 1899 [90]. Consistent with medical trials of that era, Lyonnet and his colleagues first tried the proposed drug on themselves, then on 60 of their patients (three of whom were diabetic) over a period of some months. They described what might be considered today a “Phase 0” clinical trial in somewhat vague terms: 4-5 mg sodium metavanadate (before meals) every 24 h, three times per week, with
19
resulting two out of the three diabetic patients said to have obtained
a slight,
transient, lowering of sugar levels. No ill effects were noted in any of their patients. This result remained relatively unnoticed until much later in 1979 by Tolman, et al. [91] who demonstrated that a millimolar administration of sodium metavanadate to fat cells stimulated glucose uptake and inhibited lipid breakdown in a tissue-specific manner, similar to insulin. Although inorganic salts have been successful at enhancing the activity of insulin, the poor in vivo absorption and high dose requirement resulted in increased toxicity [92]. Since insulin is not orally active, great effort has therefore been made to synthesize oxovanadium(IV) complexes of organic ligands of high biological activity (hydro/lipophilicity) and low toxicity which are readily absorbed. Potent complexes with various coordination modes VO(O4), VO(N2O2), VO(N2S2), VO(S4), VO(S2O2), and VO(N4), and the relationship between their structures and insulin-mimetic activities has been examined by evaluating both in vivo and in vitro results [93, 94].
1.5.1 Pyronates and pyridinonates Maltol (Hma) (6) and related 3-hydroxy-4-pyrones are natural products (acetogenins) which occur as fungal carbohydrate metabolites, as well as being produced as byproducts of fementation. Both maltol and ethylmaltol (7) are by themselves approved food additives in many countries. In addition, maltol is well known for formation of stable, neutrally charged metal complexes which have an optimum combination of water-solubility, reasonable hydrolytic stability, and significant lipophilicity [95-97].
Pyrones and pyridinones can act as anionic
chelating, bidentate O,O-ligands towards a number of biologically active metals [9699]. Ligands structurally related to maltol include kojic acid (Hkoj) (8), and Hdpp (1,2-
20
dimethyl-3-hydroxy-4-pyridinone) (9), both of which have substituents that can alter selectively the water-solubility, hydrolytic stability and lipophilicity of their metal complexes [98].
O
O OH O 6
O OH
O OH
O
OH
O OH
7
N 9
8
Bis(maltolato)oxovanadium(IV), BMOV or VO(ma)2 (10), consists of vanadyl ion bound to the anion of maltol (3-hydroxy-2-methyl-4-pyrone, Hma) [99]. Interest in maltol and close analogues, such as ethylmaltol (3-hydroxy-2-ethyl-4-pyrone, Hema) and kojic acid (5-hydroxy-2-hydroxymethyl-4-pyrone, Hkoj), is partly due to their ability to deprotonate readily. The geometry in the solid state around the vanadium in BMOV is square pyramidal with the two maltolato ligands in a trans arrangement around the base of the square pyramid and the V=O unit axial.
O
O
CH 3 O
V
O
O
O
O CH 3 10
BMOV is the most widely and intensively tested of the many proposed insulin mimetic vanadium complexes [86, 99-102]. In addition to lowering glucose- and lipid-levels in vivo, BMOV delays or prevents long-term diabetes-induced pathology (including cardiomyopathy) and attenuates hyperinsulinemia and hyperlipidemia in genetically diabetic rats. The longest residence times for vanadium in vivo following 21
oral administration of
48
V-BMOV were in bone (31 days), followed by liver (7 h) and
kidney (4 h) [101]. On average in these three tissues, vanadium uptake is 2–3 times greater after oral 48V-BMOV administration, compared to the same dose of 48VOSO4. Bis(ethylmaltolato)oxovanadium(IV), BEOV, VO(ema)2 [103], with slightly greater hydrolytic stability and lipophilicity, has longer turnover times in vivo, especially in bone and liver. Solubility decreases only slightly, and stability to oxidation remains unchanged from BMOV. BEOV successfully completed phase I clinical trials in early 2000. The redox chemistry of BMOV demonstrates an impressive lability in oxidation and reduction [93].
1.5.2 Acetylacetonates β-Diketones and related derivatives are considered a class of very important ligands in the growth of coordination chemistry. Their complexes have been thoroughly studied. Due to the presence of two oxygen donor atoms and facile ketoenol tautomerism they easily coordinate with metal ions after deprotonating the enolic hydrogen atom and provide stable metal complexes with six-membered chelate rings. Since the synthesis and characterization of bis(2, 4-pentanedionato) oxovanadium(IV), VO(acac)2 (11), was first published in 1914 [104], the complex has been used extensively as a reagent in organic synthesis [105-107]. The physical properties of VO(acac)2 have been examined by numerous workers [108-110]. A variety of derivatives, in which the terminal methyl group in the acac- ligands were substituted, have been prepared and studied [111, 112]. Such substitutions include both symmetric and asymmetric modifications of the parent acac- ligand and formation of the corresponding VO(acac)2-type complexes [104, 113]. Both the 3methyl- and 3-ethyl-2,4-pentanedionato oxovanadium(IV) complexes, VO(Me-acac)2
22
(12) and VO(Et-acac)2, (13) respectively, have been structurally characterized [114]. The vanadium atoms in these mononuclear complexes lie in distorted square pyramidal coordination environments. Both compounds have oxygen atoms coordinating in the equatorial plane; apical coordination by the oxo group completes the square pyramidal geometry in each case. As with the parent compound, VO(acac)2, X-ray structural analysis shows that the vanadium atom lies above the basal plane. O R
O
O
V O
R O
R= H 11, R= CH3 12, R= CH 2CH3 13.
In an in vitro study of VO(acac)2, 5–100 μM was found to be more effective than vanadyl sulphate in stimulating lipogenesis in isolated fat cells, and had identical effectiveness in stimulating activity of a cytosolic protein kinase [115]. Intraperitoneal injection (25 μmol kg−1) of VO(acac)2 lowered slightly plasma glucose levels in STZ-diabetic rats, though not to normal glucose levels; VO(Etacac)2, at the same dose, was ineffective [100]. 0.4 mM of BMOV, VO(acac)2, and VO(Etacac)2, administererd orally , were equally (mildly) effective in glucose-lowering when given in the drinking water over an 8-week treatment period, but were significantly different from VOSO4 at the same dose. In fact, the only clearly relevant physiological difference in this comparative group was between the ratio of vanadium intake/plasma vanadium levels, with VOSO4 having a remarkably higher ratio compared to all oxovanadium(IV) complexes tested [100].
23
1.5.3 Picolinates Several vanadium(IV) complexes with a VO(N2O2) chromophore have been proposed
as
insulin
mimics.
One
example
is
bis(pyridine-2-carboxylato)
oxovanadium(IV), VO(pic)2 (14), the synthesis of which was first reported in 1964 [116],
but which was only recently characterized structurally [117] and tested
biologically [117, 118]. VO(pic)2 is slightly soluble in water and as an aqueous solution in an aerobic atmosphere, is susceptible to gradual oxidation. A methyl analogue of VO(pic)2, bis(6-methylpyridine-2-carboxylato)oxovanadium(IV), VOMPA, has also been synthesized and characterized biologically, potentiometrically, and by a new blood circulation monitoring-EPR method (BCM-ESR) [119-121].
O
O
O N
V
N O
O
14
The insulin enhancing effects of picolinato chelates of oxovanadium(IV) have been clearly shown to be dependent on dose as well as delivery method. VO(pic)2, administererd at 0.2 mmol kg−1 orally for 2 days and then at 0.1 mmol kg−1 for 11 days, normalized plasma glucose in STZ-diabetic rats, a model of insulin-dependent diabetes (IDDM) [122]. Plasma insulin levels increased significantly during this trial [122]. By comparison, when VO(pic)2 was given to STZ-diabetic rats as a 2.4 mM solution (drinking water substitute), the calculated dose averaging 1.0 mmol kg−1 d−1 was accompanied by consistent glucose-lowering and no insulin elevation, but with considerable evidence of gastrointestinal irritation [117]. Intraperitoneal (i.p.)
24
administration of VO(pic)2 at doses of 0.2, 0.1, and 0.06 mmol V kg−1 d−1 [117, 122] also lowered plasma glucose levels, accompanied by increased bilirubin at the highest dose. In comparison to its methylpicolinate analogue, the picolinate complex had a less sustained response and was less effective as an inhibitor of FFA release in vitro; thus VOMPA was chosen for continued investigation [119, 121, 122]. On the other hand, comparing VO(pic)2 with BMOV [117], the picolinate complex had lower solubility and more gastrointestinal irritation for an equivalent dose, suggesting that there is room for further structural improvement in order to increase bioavailability and lessen side effects.
1.5.4 Oxovanadium(IV) salen-type derivatives Several vanadium complexes of the tetradentate Schiff base ligand N,N'bis(salicylidene)ethylenediamine (salen) (15) have been proposed for potential use as insulin mimetic agents [123]. These ligands are of particular interest because they provide coordination environments which efficiently stabilize different oxidation states of vanadium, while still providing active sites capable of binding other molecules. In addition these complexes exhibit catalytic reactivity towards organic substrates. Oxovanadium(IV) complexes have been shown to catalyze a variety of reactions such as the oxidation of alcohols and the conversion of sulphide to sulphur oxides and sulfones [124, 125]. Most oxovanadium(IV) complexes with a tetradentate Schiff base ligands like salen take green monomeric structures with square-pyramidal coordination geometry or orange linear chain structures (···V=O···V=O···) with distorted octahedral coordination in the solid state [81].
25
N
O V
N
O
O 15
To date, only [VO(sal2-en)] [126] and [VO(sal2-1,3-pn)] [127] have been tested for insulin mimetic activity from among these complexes. Unlike most biological testing for new anti-diabetic agents, this was carried out in alloxan-diabetic rats, in which blood glucose levels decreased from hyperglycemic to hypoglycemic during oral intubation of [VO(salen)] (0.15 mmol V kg−1 d−1 for 30 days). Withdrawal of treatment brought an immediate reversion to hyperglycemia. Blood glucose levels were reduced over the treatment period; liver hexokinase activity was restored, and other carbohydrate metabolism enzymes in liver and kidney were normalized. On the other hand [VO(sal2-1,3-pn)] normalized glucose and lipid values without an increase in insulin levels, and improved glucose tolerance.
1.5.5 Dicarboxylate ester–oxovanadium(IV) complexes This class of complexes was investigated as models for the interaction of vanadium(IV) with bioligands. Complexes of oxovanadium(IV) with a series of dicarboxylate ligands (oxalate, glutarate, succinate, malonate) proved effective orally as insulin mimetic agents [128]. Only the cysteine methyl ester–oxovanadium(IV) complex, VCys, was characterized by X-ray structural analysis [129]. The oxovanadium(IV) in VCys appeared to be penta-coordinate around the central vanadium atom, thus assuming a square pyramidal geometry with two-fold symmetry and each pair of nitrogen and sulphur atoms mutually trans. Insulin mimetic activity of these complexes at the doses for bioactivity (0.06 and 0.20 mmol V kg-1) showed that VCys and the bis ligand analogues of malonate, 26
tartarate, and salicylaldehyde were indistinguishable from one another in terms of glucose-lowering ability in STZ-diabetic rats. The higher dose appeared to be significantly more effective than the lower dose in all cases. An oxalic acid analogue, bis(oxalato)oxovanadate(IV) dianion, was less effective than the others; significant glucose-lowering was seen only at the 0.20 mmol V kg−1 dose level [128].
1.6 Other metal complexes used as insulin enhancing agents
1.6.1 Zinc compounds Zinc appears to play a role in modulating insulin receptor tyrosine kinase activity in the skeletal muscle of a genetic type 2 DM model mouse, similar to the action of vanadium [130]. In addition, zinc was proposed to affect carbohydrate metabolism through the insulin receptor, PTP1B, and other related proteins [131]. In fact, zinc and diabetes intersect at several points during metabolism in a cell [132, 133]. In 1980, zinc was found to stimulate rat adipocyte lipogenesis similar to the action of insulin [134], which was followed by the observations on in vivo antidiabetic effects of oral ZnCl2 in STZ-rats and ob/ob mice in 1992 [135] and 1998 [136], respectively. Because the bioavailability of ZnCl2 is relatively low, the coordination chemistry of zinc(II) ion was explored, and the first orally active insulin-mimetic and antidiabetic zinc(II)–picolinate complexes were discovered in 2002 [137]. Since then, a wide variety of zinc(II) complexes with different coordination structures have been synthesized [132, 138]. The determination of log β for zinc(II) complexes made it possible to discuss the relationship between the stability constant and the insulinmimetic activity, e.g., zinc(II) complexes with log β lower than 10.5 in vitro exhibited higher insulin-mimetic activities than those of ZnSO4 which was used as a control
27
[139, 140]. Similar to the vanadyl complexes, zinc(II)–picolinate complexes, such as [Zn(pic)2] [141], [Zn(6-mpic)] [141], and bis(6-methylpicolinemethylamido)zinc(II) [Zn(6-mpa-ma)2] [142], were prepared. The single X−ray crystal of [Zn(6mpic)2(H2O)]·H2O revealed the coordination geometry around the zinc(II) ion to be a distorted trigonal bipyramidal structure. Zinc(II)–picolinate complexes with log β lower than 10.5 exhibit higher in vitro insulin-mimetic activities than those of ZnSO4 and VOSO4. And [Zn(pic)2] with a log β = 9.52 showed higher activity than those of ZnSO4 and VOSO4. Based on the in vitro evaluation, both [Zn(pic)2] and [Zn(6mpic)2] exhibited high hypoglycemic effects in KK-Ay mice that were subjected to a single ip injection and daily ip injections at a dose of 3.0 mg Zn kg-1 body weight for two weeks [137].
1.6.2 Magnesium compounds The relationship between magnesium and diabetes has been studied for decades but it is not yet fully understood. Magnesium plays an important role in carbohydrate metabolism. It may influence the release and activity of insulin. Low blood levels of magnesium (hypomagnesemia) are frequently seen in individuals with Type 2 diabetes. Hypomagnesemia may worsen insulin resistance, a condition that often precedes type 2 diabetes, or may be a consequence of insulin resistance. Studies suggest that a deficiency in magnesium may worsen blood glucose control in Type 2 diabetes. It is believed that a deficiency of magnesium interrupts insulin secretion in the pancreas and increases insulin resistance in the body's tissues. Evidence suggests that a deficiency of magnesium may contribute to certain diabetes complications. A recent analysis showed that people with higher dietary 28
intakes of magnesium (through consumption of whole grains, nuts, and green leafy vegetables) had a decreased risk of Type 2 diabetes. Supplementation with magnesium in patients of diabetes enhances insulin sensitivity and secretion. In rats predisposed in diabetes, supplementation with oral magnesium diminished the progression of the disease. Magnesium supplements can frequently overcome several serious blood pressure disorders as well. Magnesium supplements can be available in several varieties of salts, like magnesium citrate, magnesium gluconate or magnesium lactate. 1.6.3 Chromium compounds The benefit of added chromium for diabetes control has been studied and debated for several years. Several studies report that chromium supplementation may improve diabetes control. Chromium is needed to make glucose tolerance factor, which helps insulin improve its action. The first chromium compound that has been used in the management of diabetes is chromium picolinate [143]. Studies carried out to investigate the carbohydrate metabolism in lean and obese rats when given chromium picolinate showed that the obese rats had more significant improvement in fasting insulin levels and glucose tolerance, but the lean rats did not experience any significant changes. The final conclusion was that the chromium picolinate may be beneficial in insulin resistance states but it appears to have no effects on the lean rats. The effectiveness of chromium picolinate is highly pronounced in pregnant women (gestational diabetes) and Type 2 DM. Other chromium compounds that have been tried are chromium chloride and chromium nicotinate. Because of insufficient information on the use of chromium to treat diabetes, no recommendations for supplementation yet exist.
29
1.7 Toxicity of vanadium compounds Research carried out by many workers in different laboratories [144-146] have shown that vanadium toxicity depends on specific chemical form, oxidation state, administration route, period and doses, as well as types of organism studied. The concentration limit for which vanadium compounds become toxic also depends on the type of coordination environment. As a general rule, concentration below 1.0 × 10−5 M are estimated to be safe and still are able to maintain the biological activity, whereas those above 1.0 × 10−3 M are expected to be toxic for chronic use. The chronic toxic strength patterns of vanadium compounds follow the order: inorganic salts; soluble > insoluble > organic vanadium compounds; vanadium(V) organic compounds > vanadium(IV) organic compounds. In the case of vanadium organic compounds, the toxicity also depends on the donor atoms coordinated to vanadium; NN, OO, or NO are less toxic than compounds with donor sets NS, OS, or SS, irrespective of the vanadium oxidation state. The toxicity tends to decrease as the valence decreases. Typical clinical manifestations for acute toxicity are light diarrhea, vomiting, abdominal cramps, green tongue, severe bronchospasm, neurological and irreversible renal excretion damage [147].
1.8 Accumulation of vanadium compounds in the body Vanadium tissue accumulation represents the major concern about vanadium use in the long-time administration [144]. It is noticeable that vanadium is stored in various organs with long half-lives in the body and its prolonged presence may potentially maintain some anti-diabetic activity [148]. Vanadium inorganic salts are not well absorbed by human organism; roughly 5 % of the ingested mass and only in certain condition can exceed 10 % [149]. Most ingested vanadium is apparently 30
unabsorbed and is thus excreted via the feaces. The distribution of vanadium in the body tissues after oral and intra-peritoneal administration follows the order: bone > kidney > spleen > heart > teste > lung > pancreas > brain after 24 h [150]. There is evidence that oxovanadium(IV) interacts with bone mineral, hydroxyapatite, but it is now known that the ions do not incorporate into the apatitic lattice and does not appear to affect bone strength or architecture [101]. On the other hand, vanadium organic complexes accumulation follows a pattern that is not quite different from inorganic salts. The principal sites of accumulation are bone, kidney and liver. However, the absorption of these compounds is highly improved, their retention time in plasma is also longer and their biological effects enhanced. Distribution and accumulation of vanadium is not due to increase in absorption, but depends strongly on the chemical structure [149].
1.9 Mechanism of action of insulin Insulin is a hormone which is secreted by a group of cells within the pancreas called Langerhans’ islets. It is a peptide hormone which regulates fat and carbohydrate metabolism. Insulin also serves to counteract catabolic hormone and suppress glucose production in the liver. The insulin receptor is tyrosine kinase embedded in the plasma membrane. It is composed of two alpha sub-units and two beta sub-units linked by disulfide bonds. The alpha chains house insulin binding domains while beta chains penetrate through the plasma membrane. The normal uptake and metabolism of glucose is initiated by a series of intracellular reaction known as the insulin-signaling cascade [151]. As insulin docks to the outside of the receptor, tyrosine at the inside is phosphorylated (b in Fig. 1.2), giving rise to a complicated signal transduction cascade (dashed arrow), which stimulates glucose
31
intake by a glucose carrier, symbolised by an “opening door”. This phosphorylation is counteracted by a protein tyrosine phosphatase (PTP), which catalyses the hydrolytic rupture of the phosphoester bond. This hydrolysis is fully effective in the absence of insulin (c in Fig. 1.2) or in the case of insulin tolerance. Vanadate can enter the cell via phosphate and sulphate channels. Based on the well known fact that vanadate inhibits phosphatises [152], it has been proposed and evidenced that at least one possible mechanism works through an inhibition of PTP by vanadate, allowing the phosphoester bond (which also forms by autophosphorylation) and thus the signal transduction to remain intact; d in Fig. 1.2.
Figure 1.2 Insulin-mimetic behaviour of vanadium compounds. The scheme on the top represents a simplified signal path-way induced by insulin and vanadium, respectively [153].
Alternatively, a non-membrane protein–tyrosine kinase (PTK)
may be activated by vanadium, or vanadate itself may esterificate effectively the tyrosines of the insulin receptor [147]. 32
1.10 Types of diabetes Diabetes mellitus is a group of diseases characterized by high levels of blood glucose resulting from defects in insulin production, insulin resistance, or both. Diabetes can be associated with serious complications and premature death, but people with diabetes can take steps to manage the disease and lower the risk of complications. Type1 diabetes was previously called insulin-dependent diabetes mellitus (IDDM) or juvenile-onset diabetes. Type 1 diabetes develops when the body's immune system destroys pancreatic beta cells, the only cells in the body that make the hormone insulin that regulates blood glucose. This form of diabetes usually strikes children and young adults, although disease onset can occur at any age. Type 1 diabetes may account for 5 to 10% of all diagnosed cases of diabetes. Risk factors for Type 1 diabetes may include autoimmune, genetic, and environmental factors. Type 2 diabetes was previously called non-insulin-dependent diabetes mellitus (NIDDM) or adult-onset diabetes. Type 2 diabetes may account for about 90-95% of all diagnosed cases of diabetes. It usually begins as insulin resistance, a disorder in which the cells do not utilise insulin efficiently. As the need for insulin rises, the pancreas gradually loses its ability to produce insulin. Type 2 diabetes is associated with older age (above 40 years old), obesity, family history of diabetes, history of gestational diabetes, impaired glucose metabolism, physical inactivity, and race/demography. Type 2 diabetes is increasingly being diagnosed in children and adolescents. This is attributed to changes in eating habits. Youths tend to feed more on junk foods which contain high fat content and less fiber. 33
Gestational diabetes is a form of glucose intolerance diagnosed in some women during pregnancy. Gestational diabetes is also more common among obese women and women with a family history of diabetes. During pregnancy, gestational diabetes requires treatment to normalize maternal blood glucose levels to avoid complications in the infant. After pregnancy, 5 – 10 % of women with gestational diabetes are found to progress to Type 2 diabetes. Women who have had gestational diabetes have a 20 - 50 % chance of developing diabetes in the next 5 10 years. Other specific types of diabetes result from specific genetic conditions (such as maturity-onset diabetes of youth), surgery, drugs, malnutrition, infections, and other illnesses. Such types of diabetes may account for 1 – 5 % of all diagnosed cases of diabetes.
1.11 Prevalence of diabetes in the World The prevalence of diabetes for all age-groups worldwide was estimated to be 2.8 % in 2000 and expected to rise to 4.4 % in 2030 [154]. The total number of people with diabetes is projected to rise from 171 million in 2000 to 366 million in 2030. The major part of this numerical increase will occur in developing countries. There will be a 42 % increase (from 51 to 72 million) in the developed countries and a 170 % increase (from 84 to 228 million) in the developing countries. The greatest relative increases will occur in the Middle Eastern Crescent, sub-Sahara Africa and India. The greatest absolute increase in the number of people with diabetes will be in India. Globally, diabetes prevalence is similar in men and women but there are more women with diabetes than men (CIA World Factbook 2002).
34
Figure 1.3 Worldwide map showing the prevalence of diabetes, 2003 Source: Diabetes Atlas, International Diabetes Federation (2003). The ten countries estimated to have the highest numbers of people living with diabetes in 2000 and 2030 are listed in Table 1.1
1.12 Prevalence of diabetes in South Africa Diabetes ranks the third in South Africa after ischemic heart disease and cancer in terms of morbidity and mortality. The prevalence in adults is 4% for whites, 5.8% for blacks and 13% for Indians [155]. Based on the revised WHO criteria, South African Indians show a high prevalence rate more than twice as that seen in the blacks or whites. The high prevalence of diabetes in South African Indians could be due to genetic susceptibility coupled with obesity.
35
Table 1.1 List of countries with the highest numbers of estimated cases of diabetes for 2000 and 2030 [154] Ranking
2000
2030 Country People with Country People with Diabetes (millions) Diabetes (millions) India 31.7 India 79.4 China 20.8 China 42.3 US 17.7 US 30.3 Indonesia 8.4 Indonesia 21.3 Japan 6.8 Pakistan 13.9 Pakistan 5.2 Brazil 11.3 Russian Federation 4.6 Bangladesh 11.1 Brazil 4.6 Japan 8.9 Italy 4.3 Philippines 7.8 Bangladesh 3.2 Egypt 6.7
1 2 3 4 5 6 7 8 9 10
International Diabetes Federation (IDF) atlas reports a prevalence figure of 3.4% for 24 million South Africa between the age group 20-79 (2003) with an expected increase to 3.9% by 2025 as shown in Table 1.2. It is possible that the greater degree of obesity in females could account for the somehow higher prevalence of diabetes seen in females South African. Table 1.2 Prevalence estimates of diabetes in South Africa: comparison of years 2003 and 2025 Year
Prevalence
Distribution per population/1000
(20-79 y) No.
%
Rural
Urban Male
Female 20-39
40-59 60-79
Total
2003 24 741 3.4 272.1 569.1
322.7 518.5
127.1
489.6
224.5
841.2
2025 26 816 3.9 249.3 805.7
416.8 638.2
130.2
536.3
388.5
1 055.0
Source: Diabetes Atlas, International Diabetes Federation (2003).
36
1.13 Scope of this work The scope of this work was to synthesise a series of novel symmetrical and unsymmetrical
tetradentate
Schiff
bases
derived
from
condensation
of
salicylaldehyde, substituted salicylaldehydes, 2-hydroxy-1-naphthaldehyde, and a series of aliphatic and aromatic diamines and to coordinate them with VO2+, Co2+, Ni2+, and Cu2+. The oxovanadium(IV) complexes would then be further investigated for their insulin-enhancing properties when administered as therapeutic agents.
1.14 Aims and Objectives The aim of this research work will be directed toward the synthesis and characterisation of neutral symmetrical and unsymmetrical tetradentate Schiff base complexes of oxovanadium(IV) with high thermodynamic stability, adequate balance of hydrophilicity/lipophilicity and low toxicity that could be tested in vitro and in vivo for enhancement in the activity of insulin in lowering blood glucose levels. The work would also be extended to the synthesis and physicochemical characterisation of the cobalt(II), nickel(II), and copper(II) complexes of the unsymmetrical Schiff bases only because of the extensive attention that their symmetrical Schiff base complexes have attracted in the literature [39, 46, 51].
Specifically, the research objectives are: (1)
To synthesize and characterize a series of symmetrical and unsymmetrical tetradentate Schiff bases of the N2O2 donor sets, which are anticipated to provide stereochemical flexibility and stability to their metal(II) complexes.
(2)
To coordinate the preformed ligands to the VO2+ and the unsymmetrical ligands to Co2+, Ni2+ and Cu2+ ions.
37
(3)
To establish the purity of the metal complexes and establish their stereochemistry.
(4)
To screen the oxovanadium(IV) complexes as potential insulin-enhancing agents.
1.15 Compounds reported in the project The compounds investigated in this project are listed, with their formulae, in Tables 1.3 and 1.4.
38
Table 1.3 Nomenclature and formulae for the Schiff bases Name of ligand
Formulae
N-(naphthalidene)-N'-(5-chlorosalicylidene)orthophenylenediamine
H2(naph-Clsal)opd
N-(naphthalidene)-N'-(3-ethoxysalicylidene)orthophenylenediamine
H2(naph-Oetsal)opd
N-(naphthalidene)-N'-(5-nitrosalicylidene)orthophenylenediamine
H2(naph-NO2sal)opd
N-(naphthalidene)-N'-(salicylidene)orthophenylenediamine
H2(naph-sal)opd
N,N'-bis(benzophenylidene)ethylenediamine
H2bp2en
N,N'-bis(benzophenylidene)-1-methylethylenediamine
H2bp2pn
N,N'-bis(benzophenylidene)trimethylenediamine
H2bp2tn
N,N'-bis(naphthalidene)ethylenediamine
H2naph2en
N,N'-bis(naphthalidene)trimethylenediamine
H2naph2tn
N,N'-bis(naphthalidene)orthophenylenediamine
H2naph2opd
N,N'-bis(5-chlorosalicylidene)ethylenediamine
H2Clsal2en
N,N'-bis(5-chlorosalicylidene) )-1-methylethylenediamine
H2Clsal2pn
N,N'-bis(5-chlorosalicylidene) trimethylenediamine
H2Clsal2tn
N,N'-bis(5-chlorosalicylidene) orthophenylenediamine
H2Clsal2opd
N,N'-bis (5-methoxysalicylidene) ethylenediamine
H2Omesal2en
N,N'-bis(5-methoxysalicylidene) trimethylenediamine
H2Omesal2tn
N,N'-bis(5-methoxysalicylidene) orthophenylenediamine
H2Omesal2opd
39
N,N'-bis (3-ethoxysalicylidene)ethylenediamine
H2Oetsal2en
N,N'-bis (3-ethoxysalicylidene)-1-methylethylenediamine
H2Oetsal2pn
N,N'-bis (3-ethoxysalicylidene)orthophenylenediamine
H2Oetsal2opd
Table 1.4 Nomenclature and formulae for the oxovanadium(IV) complexes Ref. No. VOL1
Name of complex
Formulae
N-(naphthalidene)-N'-(5-chlorosalicylidene)orthopheny lenediiminato oxovanadium(IV) N-(naphthalidene)-N'-(5nitrosalicylidene)orthophenylenediiminato oxovanadium(IV) N-(naphthalidene)-N'-(3ethoxysalicylidene)orthophenylenediiminato oxovanadium(IV) N-(naphthalidene)-N'-(salicylidene)orthophenylenediiminato oxovanadium(IV)
[VO(naph-Clsal)opd]
VOL5
N,N'-bis(benzophenone)ethylenediiminatooxovanadium(IV)
[VObp2en]
VOL6
[VObp2pn]
VOL7
N, N'-bis(benzophenone)-1,2-propylenediiminato oxovanadium(IV) N,N'-bis(benzophenone)trimethylenediiminatooxovanadium(IV)
[VObp2tn]·MeOH
VOL8
N,N'-bis(naphthalidene)ethylenediiminatooxovanadium(IV)
[VOnaph2en]
VOL9
N,N'-bis(naphthalidene)trimethylenediiminatooxovanadium(IV)
[VOnaph2tn]
VOL2 VOL3 VOL4
[VO(naphNO2sal)opd] [VO(naphOetsal)opd] [VO(naph-sal)opd]
VOL10 N,N'-bis(naphthalidene)orthophenylenediiminato oxovanadium(IV)
[VOnaph2opd]
VOL11 N,N'-bis(5-chlorosalicylidene)ethylenediiminatooxovanadium(IV)
[VOClsal2en]
VOL12 N,N'-bis(5-chlorosalicylidene) )-1,2-propylenediiminato oxovanadium(IV)
[VOClsal2pn]
VOL13 N,N'-bis(5-chlorosalicylidene)trimethylenediiminato oxovanadium(IV) VOL14 N,N'-bis(5-chlorosalicylidene)orthophenylenediiminato oxovanadium(IV)
[VOClsal2tn]
40
[VOClsal2opd]
VOL15 N,N'-bis (5-methoxysalicylidene)ethylenediiminato oxovanadium(IV) VOL16 N,N'-bis (5-methoxysalicylidene)trimethylenediiminato oxovanadium(IV) VOL17 N,N'-bis (5-methoxysalicylidene)orthophenylenediiminato oxovanadium(IV)
[VO(Omesal2en)]
VOL18 N,N'-bis (3-ethoxysalicylidene) ethylenediiminato oxovanadium(IV) 19 VOL N,N'-bis (3-ethoxysalicylidene) )-1,2-propylenediiminato oxovanadium(IV) VOL20 N,N'-bis (3-ethoxysalicylidene) )orthophenylenediiminato oxovanadium(IV)
[VO(Oetsal2en)]
CoL1
N-(naphthalidene)-N'-(5-chlorosalicylidene)orthopheny lenediiminato cobalt(II) N-(naphthalidene)-N'-(5nitrosalicylidene)orthophenylenediiminato cobalt(II) N-(naphthalidene)-N'-(3-ethoxysalicylidene) orthopheny lenediiminato cobalt(II) N-(naphthalidene)-N'-(salicylidene)orthophenylenediiminato cobalt(II)
[Co(naph-Clsal)opd]
N-(naphthalidene)-N'-(5-chlorosalicylidene)orthopheny lenediiminato copper(II) N-(naphthalidene)-N'-(5-nitrosalicylidene)orthopheny lenediiminato copper(II) N-(naphthalidene)-N'-(3-ethoxysalicylidene)orthopheny lenediiminato copper(II) N-(naphthalidene)-N'-(salicylidene)orthophenylenediiminato copper(II)
[Cu(naph-Clsal)opd]
N-(naphthalidene)-N'-(5-chlorosalicylidene)orthopheny lenediiminato nickel(II) N-(naphthalidene)-N'-(5nitrosalicylidene)orthophenylenediiminato nickel(II) N-(naphthalidene)-N'-(3-ethoxysalicylidene)orthopheny lenediiminato nickel(II) N-(naphthalidene)-N'-(salicylidene)orthophenylenediiminato nickel(II)
[Ni(naph-Clsal)opd]
CoL2 CoL3 CoL4
CuL1 CuL2 CuL3 CuL4
NiL1 NiL2 NiL3 NiL4
41
[VO(Omesal2tn)].MeOH [VO(Omesal2opd)]
[VO(Oetsal2pn)] [VO(Oetsal2opd)]
[Co(naphNO2sal)opd] [Co(naphOetsal)opd] [Co(naph-sal)opd]
[Cu(naphNO2sal)opd] [Cu(naphOetsal)opd] [Cu(naph-sal)opd]
[Ni(naphNO2sal)opd] [Ni(naphOetsal)opd] [Ni(naph-sal)opd]
CHAPTER 2
PHYSICAL TECHNIQUES USED FOR CHARACTERIZATION
2.1 Electron paramagnetic resonance (EPR) EPR, also called electron spin resonance (ESR), is a form of magnetic resonance spectroscopy used for measuring the absorption of electromagnetic radiation by a molecular system containing one or more unpaired electrons [156, 157]. The sample is usually placed in a magnetic field and the transitions monitored are between two electron energy levels. Most experiment uses 9-9.5 GHz microwaves radiation in the X-band, for which the free electron resonance occurs at about 3200-3400 G. This sensitive technique has proved useful in the study of the electronic structures of many species, including organic free radicals, biradicals, triplet excited states and most transition metals and rare-earth metals. Important biological applications include the use of 'spin labels' as probes of molecular environment in enzyme-active sites and membranes [156]. EPR has also been used to examine interior defects in solid state chemistry and to study reactive chemical species on catalytic surfaces. EPR gives the chemical information regarding the structure of paramagnetic substances. The number of lines, their spacing and their relative intensities unequivocally indicate a characteristic structure of a species. It is observed that when a molecule or ions containing one or more unpaired electrons are placed in a magnetic field, the effect of the magnetic field is to lift the spin degeneracy, i.e., to make the energy of the electrons differ for its two Ms values, +½ and -½. The electron lines up its field with the magnetic field and results in an increase of 42
potential energy. A quantum-mechanical treatment shows that the energy difference between these two electron spin alignments is equal to g H, where g is the gyromagnetic ratio,
is the Bohr magneton and H is the strength of magnetic field.
For EPR investigation, transition metal ions are the simplest example. To understand the spectrum of an ion requires a detailed consideration both of the individual ion and of its environment. The result may provide: i) Identification of the elements, its specific valence state and composition; ii) The symmetry of the crystalline electric field to which an ion is subjected, and iii) Numerical values for parameters in spin Hamiltonian. When the ions are placed in condensed media, their behavior in a magnetic field is profoundly altered. In evaluation of an EPR spectrum, the most important parameter is the g value, which is also known as spectroscopic splitting factor.
2.1.1 The theory of EPR spectroscopy [157] The theory of EPR spectroscopy shares much in common with that of nuclear magnetic resonance spectroscopy (NMR). However, the magnetic moment of the electron is about 1000 times as large as the nuclear moment and the constants employed in NMR theory frequently are different in magnitude and sign. The electron is a charged particle with angular momentum (orbital and spin) and as such, it possesses a magnetic moment, μe, given by e=
g J -------------------------------------------------------------Equation 1
Here J (in units of h/2π) is the total angular momentum vector, h = Planck’s constant, g is a dimensionless constant (the g-values, g-factor, or spectroscopic splitting factor), and
is a constant, the Bohr magneton. The negative sign in Eq. 1 is a
43
consequence of negative electronic charge. Neglecting orbital angular momentum and considering only the total spin angular momentum S, Eq. 1 can be written as e=
g S -----------------------------------------------------------Equation 2
The g-value for the free electron, ge, is 2.0023. The approximation made in Eq. 2 is valid for most discussions of the EPR spectra of transition-metal complexes whose orbital angular momentum can be considered to be ‘’quenched’’. Treating the g-value as an experimental quantity does not harm the present discussion, since deviation of g-values from ge can be accounted for by introduction of spin-orbital coupling. Magnetic moments can be detected by their interactions with magnetic fields. In zero field, the magnetic moments of unpaired electrons in a sample are randomly oriented. In the presence of a magnetic field, it gives rise to 2S+1 energy states (Zeeman splitting). The measurable components of μe are g ms where ms is the magnetic spin quantum number, which can take the values +S, +(S 1),....,
(S
1),
-S. The application of a magnetic field to an S=½ or larger system is said to remove the spin degenerancy. The energy of an electron moment in a magnetic field is given by E=
eH
------------------------------------------------------------------Equation 3
Combining Eq. 2 and 3, the expression E = g ms -------------------------------------------------------------------Equation 4 where S = ±½, yields two energy levels Ems = +1/2 = +½ g H -------------------------------------------------------Equation 5 and Ems = -1/2 =
½ g H ------------------------------------------------------Equation 6
whose energy is linearly dependent on H. The separation between these energy levels at a particular value of the magnetic field, H is 44
E = +½ g HR
( ½ g HR) = g H ------------------------------Equation 7
In an EPR experiment, an oscillating magnetic field perpendicular to the H induces transition between the ms =
½ and ms = +½ levels, provided the frequency, ν, is
such that the resonance condition, E = hν = g H --------------------------------------------------------Equation 8 is satisfied. The frequency is held constant and the magnetic field is varied. At a particular value of the magnetic field, H, resonance absorption of energy occurs, resulting in a peak in the spectrum (Fig. 2.1).
ms = + 1 /2
Energy
−
E = g βΗ
0
+
ms = - 1/ 2
Magnetic field
Fig. 2.1 Energy levels for an unpaired electron in a magnetic field
The EPR spectrum of oxovanadium(IV) complexes consists of eight-line signal (2I+1) arising from the interaction of a single unpaired electron (S =½) with the quenched orbital angular momentum of vanadium nucleus of spin I = 7/2 [158, 159].
45
2.1.2 The Effects of Point Symmetry The point symmetry at the metal determines whether or not any of the principal values of g or of hyperfine splitting constant (A) are required to be equal to each other. Also it determines whether or not any of the principal axes of g and A are required to be coincident. These criteria are summarized in Table 2.1, along with the accepted nomenclature for EPR behaviour and their associated point symmetries. The importance of these relationships is that each type of EPR behaviour is associated with a restricted number of point symmetries. This in turn places constraints upon the geometrical structures of the paramagnet [160].
Table 2.1 Relationships between g and A tensors, EPR symmetry and the point symmetry of paramagnets [160] EPR Symmetry
g and A Tensors
Coincidence of Tensors Axes
Molecular Point Symmetry
Isotropic
gx= gy = gz
All coincident
Oh, Td, O, Th, T
All coincident
D4h, C4v, D4, D2d,
All coincident
D6h, C6v, D6, D3h, D3d, C3v, D3 D2h, C2v, D2
Ax = Ay = Az Axial
Rhombic Monoclinic Triclinic Axial Non-collinear
gx= gy
gz
Ax = Ay
Az
gx gy Ax Ay gx gy Ax Ay gx gy Ax Ay gx= gy Ax = Ay
gz Az gz Az gz Az gz Az
One axis of g and A coincident Complete non-coincident Only gx and Az coincident
46
C2h, Cs, C2 C2, C1 C3, S6, C4, S4 C4h, C6, C3h, C6h
2.2 Magnetic susceptibility For studying the electronic structure of a transition metal complex, the measurement of magnetic moment is a very useful method. It provides fundamental information about the bonding and stereochemistry of metal complexes. The magnetic properties of coordination compounds are based on the effect of ligands on the spectroscopic terms of metal ions [161]. The Gouy method is the simplest method of measuring magnetic moments. It consists of a suspension of a uniform rod in a nonhomogeneous magnetic field of about 5000 Oersteds and measuring the force exerted on it by a conventional weighing technique. The caliberants usually used are Hg[Co(SCN)4] and [Ni(en)3S2O3] which are easy to prepare, do not decompose or absorb moisture and pack well in the sample tube. Their susceptibilities at 20 °C are; 16.44 x 10-6 and 11.03 x 10-6 c. g. s. units, respectively and may decrease from 0.05 x 10-6 to 0.04 x 10-6 per degree rise in temperature. All substances possess magnetic properties and are affected by the application of a magnetic field [162]. The substances may be diamagnetic when an apparent reduction in mass is caused in the applied magnetic field and paramagnetic when an apparent increase in mass is caused in the magnetic field. The molar susceptibility, a measure of magnetic field, of a substance is an algebraic sum of the susceptibilities of the constituent atoms, ions or molecules. The susceptibility per gram atom of a paramagnetic metal ion in a particular compound is determined by measuring the molar susceptibility of the compounds and applying diamagnetic corrections for the other ions or molecules in the compound. The diamagnetic corrections can be estimated by various methods [163]; Pascal's correction gives satisfactory results for inorganic compounds.
47
2.2.1 The theory of magnetic susceptibility [162, 164] When a substance is placed in a magnetic field of strength H, the magnetic induction or density of lines of force, B, within the substance is given by: B = H + 4 I --------------------------------------------------------Equation 9 where I is the intensity of magnetization or magnetic moment per unit volume and the term 4 I is a contribution to B by the substance itself. Dividing eq. 9 by H gives: P = 1 + 4 κ -----------------------------------------------------Equation 10 where P and κ are the magnetic permeability of the material and the magnetic susceptibility per unit volume respectively, which may be considered dimensionless. Thus the volume susceptibility of a vacuum is zero, since in a vacuum B/H = 1. The volume susceptibility of a diamagnetic substance is negative while paramagnetic substances have positive susceptibilities. In practice, susceptibility is usually expressed per unit mass (gram susceptibility) rather than per unit volume. χg = κ/ ---------------------------------------------------------Equation 11 where χg = gram susceptibility = density of the substance in gcm−1 χm = χgM -------------------------------------------------------Equation 12 where M is the molar mass of the sample. Similarly, χa and χa+ refer to the atomic and ionic susceptibilities respectively and can be obtained from equation 13. χa = χm
ΣχL ------------------------------------------------Equation 13
where χL is the molar susceptibility of all other constituents of the ligands (diamagnetic correction). The magnetic moment is given by μeff = 2.828 χaT BM ---------------------------------Equation 14 48
where T is the temperature in K. Experimental values of χa are very small and are generally independent of both field strength and temperature.
2.2.2 Types of magnetic behaviour If P 1 (i.e. I, κ and χ are negative) the substance is said to be diamagnetic. It causes a reduction in the intensity of the magnetic field and in an inhomogenous field moves to the region of lowest field strength. The molar susceptibility of diamagnetic substances is very small and negative (-1 to 100
10-6 c.g.s. e.m.u)
and are usually independent of field strength and temperature. Diamagnetism is a property of all matter and arises from the interaction of paired electron with the magnetic field. Diamagnetic susceptibilities of atoms in molecule are additive; and this is of particular use in estimating the diamagnetic susceptibilities of ligand atoms and counter-ions in a transition metal complex.[162] Additivity of atomic susceptibility is contained in the Pascal’s constants. Paramagnetism results when P 1 i.e. I, κ and χ are positive, and it causes an increase in the intensity of the field and in an inhomogenous field it moves to the region of highest fields strength. Paramagnetic susceptibilities are positive and relatively large (100 to 100, 000
10-6 c.g.s. e.m.u). They are independent of field
strength but depend inversely as temperature. However, temperature independent paramagnetism (TIP) can arise in system containing unpaired electron due to the coupling of the ground states with the excited states under the influence of a magnetic field. Ferromagnetism and anti-ferromagnetism are both special classes of paramagnetism. They arise from the interaction of individual paramagnetic species with one another. In anti-ferromagnetism, the magnetic vectors of the neighbouring 49
centres tend to couple anti-parallel so as to cancel one another. It reduces the susceptibility and hence magnetic moment of a compound while in ferromagnetic substances the moments of the separate ions tend to align themselves parallel and thus to reinforce one another. They are both temperature and field strength dependent [161].
2.2.3 Magnetic properties of transition metal complexes For ions of the first transition series, the magnetic moments due to electron spin (spin only moment) is given by the formula: μs.o = [4S(S+L)]½ -----------------------------------------Equation 15 where S = sum of the spin quantum numbers, s =
½ , hence the number of
unpaired electrons n = 2S Equation 15 can therefore be rewritten as: μs.o = [n(n+1)]½ -------------------------------------------Equation 16 Where there is orbital motion, the magnetic moment can be written as: μS+L = [4S(S+L) + L(L+1)] ½ ---------------------------------------Equation 17
50
Table 2.2 The expected spin only moments for n unpaired electron system
No. of unpaired electron
μs.o (B.M)
S
1
1.7
½
2
2.83
1
3
3.87
4
4.90
5
5.92
6
6.39
7
7.94
3
/2
2 5
/2
3 7
/2
2.3 UV-Visible spectrophotometry The excitation of a molecule from its electronic ground state to an electronic excited state corresponds to absorption of light in the near-infrared, visible or ultraviolet regions of the spectrum. For transition metal complexes, the absorption bands in the first two of these regions (infrared and visible) are relatively weak and are associated with transitions largely localized on the metal atom. The ultraviolet bands are intense and they are associated with the transfer of an electron from one atom to another and so are called charge-transfer bands. The spectra of transition metal complexes depend on the transition of unpaired electrons from the ground state to an excited state. Transitions may occur between the split d-levels of the central atom, giving rise to the d-d or ligand field spectra. The spectra region where these bands occur spans the near infra-red, visible and UV. Most of the transition metal complexes are coloured due to d-d transitions in the visible region. The atomic overlap in metal—ligand bonds allows d 51
electrons to penetrate from the central atom to the ligand, and vice versa. The transitions are affected by the effect of ligands on the energies of the d orbital of the metal ions. Since octahedral, square-planar and tetrahedral fields cause splitting of d orbitals in different ways, the geometry will have a pronounced effect on the d-d transitions in a metal complex. Thus spectral data of transition metals provide useful information about the structure of complexes. The optical spectra of oxovanadium(IV) complexes are characterized by three d-d transitions. These transitions have been assigned using the energy level scheme by Ballhausen and Gray [87]. In this scheme, band I is assigned as b2→ eπ* (11,00016,000), band II as b2→b1* (14,500-19,000) and band III as b2→ a1* (21,00030,000). An overlap between band III and charge transfer bands has always been a problem in being able to make a definitive assignment of band III. It tends to ‘borrow’ intensity from a nearby charge transfer transition, which in many instances reduces it to a shoulder [165].
2.3.1 The Molecular Orbital Approach for oxovanadium(IV), [VO(H2O)5] 2+, ion In
an
attempt
to
developed
appropriate
energy
level
scheme
for
oxovanadium(IV) ion, earlier theory using crystal field models of the ion were proposed by Furlani and Jørgensen [32, 166]. Furlani’s calculations were restricted to C∞v symmetry of oxovanadium(IV) ion and therefore could not account for all the observed energy levels. Considering the tetragonal molecular ion, [VO(H2O)5]2+, with axial destabilization, Jørgensen obtained a scheme of energy levels which quantitatively accounted for the ‘crystal field’ part of the spectrum. The weakness of these early models was first pointed out by Palma-Vittorelli, et al. [167] and later by Ballhausen and Gray [87]. They concluded from their magnetic and spectral data 52
that a pure crystalline field model, that is, a model which only considers σ-bonding to be present, could not provide adequate description of the electronic structure of VO2+. For accurate description of the electronic structure of oxovanadium(IV) ion and its complexes, provisions have to be made for π-bonding between vanadium and oxygen. This was achieved by the molecular orbital treatment of Ballhausen and Gray [87]. Ballhausen and Gray treated the molecular ion, [VO(H2O)5]2+, in a sophisticated and detailed calculation, necessarily with many approximations. They arrived at the energy level scheme shown in Figure 2.1. The orbitals involved in bonding are the 3d, 4s and 4p of the metal, along with the 2s, 2pσ (2pz) and 2pπ (2px, 2py) of the oxygen and the spσ hybrid orbitals for the water oxygens. The crystal field transitions involve promoting the b2 electron to the eπ*, b1*, a1*, molecular orbitals which are essentially the 3d metal orbitals resulting in 2E(I), 2
B1 and 2A1 excited states respectively. The b2 level is presumed to be purely
vanadium 3dxy orbital, while the eπ* orbital is made up of a linear combination of vanadium 3dxz and 3dyz orbitals with oxygen 2px and 2py orbitals. The eπ* energy level should therefore be sensitive to any perturbation that changes the axial compression in the vanadium-oxygen bond. A strong axial perturbation would be expected to reduce the vanadium-oxygen interaction thus lowering the eπ* level with respect to b2*. The b1* level is made up of a linear combination of the 3dx2-y2 metal orbital and ligand orbitals concentrated in the equatorial plane.
It is therefore
expected that while any axial perturbation will only affect this level indirectly, it will be sensitive to electronic changes in the equatorial coordinating atoms. The a1* level involves an admixture of the 3dz2 orbitals with the metal 4s and ligand orbitals. One
53
might expect to observe shifts in the band position on trans-ligation and with changes in the solvent. The Ballhausen and Gray scheme has been used extensively by other workers to interprete optical, EPR and magnetic properties of oxovanadium(IV) complexes. However, many experimental results are being reported which tend to question the universality of the scheme. Selbin was the first to question the general applicability of the Ballhausen and Gray scheme. The basic assumptions of a C4v symmetry and an O=V— equatorial ligand angle of 90 oC were in error as X−ray measurements later showed [168, 169]. Band III was observed at 25,000 cm-1 for bis(acetylacetonato)oxovanadium( IV) which has a higher V=O bond order [170] than the same bond in [VO(H2O)5]2+. Ballhausen and Gray did not observe any band between 16,000 and 41,000 cm-1. Measurements at low temperature and on low-symmetry compounds are strengthening these doubts about the universality of the Ballhausen and Gray scheme. Selbin has suggested that the ground level is the singly occupied, nonbonding b2 orbital but that the eπ* and b1* levels may be very close in energy such that they may cross one another from complex to complex. Kolawole, et al. [40, 41] did provide experimental evidence for the inversion of eπ* and b1* in a series of symmetric Schiff base complexes of oxovanadium(IV) as the methylene bridge increases from 2 to 10 and proposed an energy level which incorporates Ballhaussen and Gray, and Selbin’s schemes.
54
e*σ
IIIa*1 IIa* 1
4p
Ia*1
4s b*1 e*π 3d
b2
(Oxide)
eπ IIIa1
(H 2O)
eσ b1 IIa1
(Oxide)
Ia 1
Fig. 2.2 Energy level ordering of Ballhausen and Gray [87]
Patel and Kolawole [41] provided an energy level that show the changes in the ordering of the d orbitals in oxovanadium(IV) complexes as shown in Fig. 2.2, which can be used to account for the electronic spectra of not only different square pyramidal complexes but also of octahedral ones.
55
z2
x2 -y 2
xz, yz
xy ~C 4v
~O h
~C 2v
~C 4v
Fig. 2.3 Changes in the ordering of the d orbitals in oxovanadium(IV) complexes
2.4 Thermal Analyses (TA) Thermal analyses refer to a group of techniques in which a property of a sample is monitored against time or temperature while the temperature of the sample, in a specified atmosphere, is programmed. Thermal analyses are based upon the detection of changes in the enthalpy or the specific heat of a sample as temperature is increased. As thermal energy is supplied to the sample its enthalpy increases and its temperature rises by an amount determined, for a given energy input, by the specific heat of the sample. The specific heat of a material changes slowly with temperature in a particular physical state, but alters discontinuously at a change of state. The supply of thermal energy may induce physical or chemical processes in the sample, e.g. melting or decomposition, accompanied by a change in enthalpy, the latent heat of fusion, heat of reaction, phase transformation, etc.
56
Such enthalpy changes may be detected by different thermal analyses and related to the processes occurring in the sample.
2.4.1 Types of TA Instrumentation Thermal analyses encompass a wide variety of techniques such as: (a) Thermogravimetric analysis (TGA) (b) Differential thermal analysis (DTA) (c) Differential scanning calorimetry (DSC) (d) Thermal mechanical analysis (TMA) (e) Temperature resolved X-ray diffraction (f) Thermomagnetometry (g) Thermoconductometry (h) Dilatometry The first three on the list are the most common types of thermal analyses used and discussion will be centred on them. Thermogravimetric analysis (TGA) is an analytical technique used to determine a material’s thermal stability and its fraction of volatile decomposition products by monitoring the weight change that occurs as a specimen is heated. The measurement is normally carried out in air or in an inert atmosphere, such as helium or argon, and the weight is recorded as a function of increasing temperature. Sometimes, the measurement is performed in a lean oxygen atmosphere (1 to 5% O2 in N2 or He) to slow down oxidation. In addition to weight changes, some instruments also record the temperature difference between the specimen and one or more reference pans (differential thermal analysis, or DTA) or the heat flow into the specimen pan compared to that of the reference pan (differential scanning
57
calorimetry, or DSC) [171]. The latter can be used to monitor the energy released or absorbed via chemical reactions during the heating process. In thermogravimetry the change in mass of the sample is recorded as a function of temperature. It provides the analyst with quantitative measurements of change in weight associated with any transition. TG can directly record the loss in weight
with
time
or
temperature
due
to
dehydration
or
decomposition.
Thermogravimetric curves are characteristic of a given compound or system because of the unique sequence of physiochemical reactions which occur over definite temperature ranges [172]. In order for a mass change to be detected, a volatile component must he evolved or absorbed by the sample. The former is the usual mode of measurement, but many examples are also known for the latter. Since elevated temperatures are normally required for the evolution of volatile materials, mass-change measurements are made at increasing rather than decreasing temperatures. Routine measurements can be made at temperature range from ambient to 1500°C under inert atmosphere. Differential thermal analysis (DTA) is a method of measuring the temperature difference between a sample and a reference sample under a controlled temperature-time programme. The instrument is composed of two identical cells in which the sample and a reference (often an empty pan) are placed. Both cells are heated with a constant heat flux, Q, using a single heater, and the temperatures of the two cells are measured as a function of time. If the sample undergoes a thermal transition such as melting, glass transition, chemical reactions, phase changes or structural changes occurring in the sample, liberation or absorption of energy by the sample with the corresponding deviation of its temperature from that of the reference is registered. The difference in temperature is represented by equation 18,
58
T = Tsample ―Treference ---------------------------------------Equation 18 Negative
T indicates an endotherm for a heating cycle. DTA curves are useful for
both quantitative and qualitative estimate of energies associated with different transformations in thermal processes. The positions and shapes of the peaks can be used to determine the changes in composition of the sample due to dehydration, decomposition and polymerization. The shape of the DTA curve can also be used in evaluating the kinetics of the reaction under carefully controlled conditions. It is important to note that thermal analysis is affected by experimental conditions and deviations caused by instrumental factors. Quantitative analysis of DTA data is complicated and the instrument is usually viewed as a fairly crude sibling of a differential scanning calorimeter (DSC) discussed below. Recent instrumental advancements have improved the quantitative use of DTA instruments. A DTA instrument is generally less expensive than a DSC. Determinations of transition temperatures are accurate in a DTA. Estimates of enthalpies of transition are generally not accurate. In the DTA heat is provided at a constant rate and temperature is a dependent parameter. Differential scanning calorimetry (DSC) is a technique in which the difference in energy inputs into a substance and a reference material is measured as a function of temperature while the substance and the reference material are subjected to a controlled temperature program. DSC is the most widely used thermoanalytical technique. It enables to determine a number of parameters connected with the physical or chemical processes in condensed phase. DSC monitors heat effects associated with phase transitions and chemical reactions as a function of temperature. In a DSC the difference in heat flow to the sample and a reference at
59
the same temperature is recorded as a function of temperature. The reference is an inert material such as alumina, or just an empty aluminum pan. The temperature of both the sample and reference are increased at a constant rate. ∆dH/dt = (dH/dt)sample – (dH/dt)reference--------------------------------Equation 19 The heat flow difference can be either positive or negative. In an endothermic process, such as most phase transitions, heat is absorbed and, therefore, heat flow to the sample is higher than that to the reference. Temperatures of phase transition, enthalpies of phase transitions, polymorphism in food and pharmaceuticals, liquid crystalline transitions, phase diagrams, thermoplastic polymer phase changes, glass temperatures, purity measurements and kinetic studies can be mentioned as examples where DSC is highly efficient.
2.5 Cyclic Voltammetry Cyclic voltammetry (CV) is perhaps the most effective and versatile electroanalytical technique available for the mechanistic study of redox systems. It enables the electrode potential to be rapidly scanned in search of redox couples [173].
Once located, a couple can then be characterized from the potentials of
peaks on the cyclic voltammogram and from changes caused by variation of the scan rate. In cyclic voltammetry, the electrode potential ramps linearly versus time. This ramping is known as the experiment's scan rate (V/s). The potential is measured between the reference electrode and the working electrode and the current is measured between the working electrode and the counter electrode. This data is then plotted as current (i) vs. potential (E). The forward scan produces a current peak for any analytes that can be reduced (or oxidized, depending on the initial scan direction) through the range of the potential scanned. The current will
60
increase as the potential reaches the reduction potential of the analyte, but then falls off as the concentration of the analyte is depleted close to the electrode surface. If the redox couple is reversible then when the applied potential is reversed, it will reach the potential that will reoxidize the product formed in the first reduction reaction, and produce a current of reverse polarity from the forward scan. This oxidation peak will usually have a similar shape to the reduction peak. As a result, information about the redox potential and electrochemical reaction rates of the compounds is obtained. For instance if the electronic transfer at the surface is fast and the current is limited by the diffusion of species to the electrode surface, then the current peak will be proportional to the square root of the scan rate. The important parameters of cyclic voltammetry are the magnitude of the peak currents, ipa and ipc, and the potentials at which peaks occur, Epa and Epc. Reversible peaks have a distinct absolute potential difference between the reduction (Epc) and oxidation peak (Epa). In an ideal system |Epc-Epa| would be 59 mV for a 1 electron process and 30 mV for a 2 electron process [174]. In addition th e ratio of the currents passed at reduction (ipc) and end oxidation (ipa) is near unity (ipa/ipc=1) for a reversible peaks. When such reversible peaks are observed thermodynamic information in the form of half cell potential E01/2 can be determined. When waves are semi-reversible such as when ipa/ipc is less than or greater than 1, it can be possible to determine even more information especially kinetic processes like following chemical reaction. When waves are non-reversible it is impossible to determine what their thermodynamic E01/2 is with cyclic voltammetry.
61
.
CHAPTER 3 EXPERIMENTAL 3.1 Materials All reagents and chemicals purchased from Aldrich-Sigma were of analytical/spectroscopic grade and used without further purification. Chemicals and solvents used for the preparation of Schiff base ligands and metal complexes are: salicylaldehyde,
5-chlorosalicylaldehyde,
5-nitrosalicylaldehyde,3-ethoxysalicyl
aldehyde, 5-methoxysalicylaldehyde, 2-hydroxy-1-naphthaldehyde, ethylenediamine, 1,2-diaminopropane, 1,3-diaminopropane, 2-hydroxybenzophenone, 1,2-phenylene diamine,
oxovanadium(IV)sulphate
hydrate,
tetrabutylammoniumperchlorate,
cobalt(II) acetate, nickel(II) acetate, copper(II) acetate,
ethanol, methanol,
triethylamine, chloroform, dimethsulphoxide, toluene, and dichloromethane.
3.2 Materials for insulin-mimetic test The following materials were used for the insulin-mimetic test: C2C12 mouse skeletal muscle cells were obtained from the American Type Culture Collection (ATCC number CRL-1772) and adult male Wistar rats (400-450g) were obtained from the Experimental Animal house of Diabetes Discovery Platform, South African Medical Research Council, Cape Town. Dulbecco modified Eagle’s medium (DMEM), L-glutamine, sodium bicarbonate and glucose were purchased from Lonza (USA) while fetal bovine serum (FBS) and horse serum were purchased from Highveld Biological (South Africa).
3.3 Synthesis 3.3.1. Preparation of unsymmetrical Schiff bases 62
Ethanolic solutions of 2-hydroxy-1-naphthaldehyde (3.444 g, 20 mmol), 1,2phenylenediamine (2.163 g, 20 mmol) and salicylaldehyde or substituted salicylaldehyde (20 mmol) in absolute ethanol (75 mL each) were prepared and chilled in the refrigerator at 4 oC for 15 min. To a stirred solution of the cold 2hydroxy-1-naphthaldehyde, cold solution of 1, 2-phenylenediamine was added dropwise followed by the addition of cold solution of salicylaldehyde (or substituted salicylaldehyde) over a period of 2 min. The mixture was kept stirred at room temperature for 4 days, after which the mixture was warmed to, and kept at, 70 oC for 20 min with stirring to dissolve any unreacted reactants and to complete the reaction. The product was filtered hot and washed twice with ice cold ethanol. The orange product obtained was purified by digesting in hot ethanol, filtered hot, and dried in a desiccator over silica gel. 3.3.2 Preparation of symmetrical Schiff bases The ligands were prepared according to established procedures [175].
A
typical procedure for the synthesis of the symmetrical Schiff base is as follows: To a stirred solution of 0.08 mole of the appropriate 2-hydroxycarbonyl compound (aldehyde and ketone) in 60 mL absolute ethanol was added, drop wise, 0.04 mol of an ethanolic solution of aliphatic or aromatic diamines. This mixture was then stirred for 2 h at 50 oC. Afterwards the mixture was cooled to room temperature, or in ice, and the products formed were collected by filtration. The crystals were washed with cold absolute ethanol and re-crystallized from ethanol-chloroform (1:3 v/v) mixture. The yellow crystals were dried in a desiccator over silica gel.
3.3.3 Preparation of oxovanadium(IV) complexes
63
The following general procedure was used in the synthesis of all the oxovanadiun(IV) complexes [41]. Oxovanadium(IV) sulphate (6 mmol, 0.978 g) was dissolved in hot absolute methanol (300 mL) and a mixture of triethylamine (12 mmol, 1.214 g) and the corresponding Schiff bases (6 mmol), dissolved in methanol (20 mL), was added with stirring, which resulted in an instant colour change to green/orange. The mixture was stirred for 3 h at 50 oC and then concentrated to half of its volume using a rotary evaporator. The product was filtered and washed twice with cold absolute ethanol and allowed to dry in a desiccator over silica gel.
3.3.4. Preparation of the cobalt(II), nickel(II) and copper(II) complexes The various complexes were prepared by addition of 3 mmol of Co(CH3COO)2.4H2O (0.53 g), Ni(CH3COO)2.4H2O (0.75 g) or Cu(CH3COO)2.H2O (0.60 g) dissolved hot absolute methanol (60 mL) to a stirring 3 mmol of the respective unsymmetrical Schiff bases in methanol (40 mL). The colour of the mixture changed instantly. The mixture was refluxed for 3 h and the precipitated solids were filtered, washed with cold methanol and allowed to dry in a desiccator over silica gel.
3.4 Characterization of the ligands and complexes The ligands were characterized by elemental analysis, infrared and 1H NMR while the metal complexes were characterized by elemental analyses, infrared, and electronic spectral, cyclic voltammetry, electron paramagnetic resonance, and room temperature magnetic susceptibility measurements, and differential scanning calorimetry.
64
3.4.1 Microanalysis Carbon, hydrogen and nitrogen analysis was done in house on a PerkinElmer automated model 2400 Series II CHNS/O analyzer.
3.4.2 Melting/decomposition points The melting/decomposition points were determined in house by placing a finely
powered
sample
in
a
glass
capillary
and
heating
by
using
Barnstead/electrothermal digital melting point apparatus and are uncorrected.
3.4.3 1H NMR spectra The 1H NMR spectra of the ligands, in CDCl3 with tetramethylsilane (TMS) as an internal standard, were obtained using a Bruker Avance III 400 MHz spectrophotometer at University of KwaZuluNatal Westville campus. All chemical shifts are given in ppm versus tetramethylsilane.
3.4.4 Infrared spectroscopy Infrared
spectra
were
recorded
on
a
Bruker
FT-IR
tensor
27
spectrophotometer directly on small samples of the compounds in the range 2004000 cm–1. The infrared spectra measurements were carried out in house.
3.4.5 Electronic absorption spectra Electronic absorption spectra in the UV-Visible region were recorded on a Cary Model 50 spectrophotometer between 200-1100 nm in both chloroform and DMSO as solvents. These measurements were also carried out in house.
65
3.4.6 Electron paramagnetic resonance (EPR) EPR spectra were measured using a Bruker EMX Micro Premium X Spectrometer at X-band (9.4 GHz) on the powder and on the fluid and frozen solutions in toluene/dichloromethane (90/10 v/v). The EPR spectra measurements were run by Dr. J Wolowska of the University of Manchester, United Kingdom.
3.4.7 Magnetic moments Magnetic susceptibility measurements done in house were made on powdered samples using a Sherwood Scientific magnetic susceptibility balance. Hg[Co(SCN)4] was used as the calibrant and corrections for diamagnetism were estimated from Pascal’s constants.
3.4.8 Cyclic voltammetry The cyclic voltammetry was run by Professor Kolawole during his sabattical visit to the Jackson State University, USA on CHI 832 electrochemical detector. Glassy carbon electrode, platinum wire and Ag/Ag+ were used as working, supporting and reference electrodes respectively. Sample solutions were 10−3 M of each complex in spectroscopic grade DMSO containing 0.1 M tetrabutylammonium perchlorate as the supporting electrolyte. Each solution was degassed with ultra pure N2 gas for 5 min before each measurement was made.
3.4.9. Differential scanning calorimetry (DSC) Thermal analysis was carried out by Professor Kolawole during his sabattical visit to the Jackson State University, USA on Netzsch Thermal Analysis DSC 200
66
F3. The sample and reference pan are in separate furnaces heated by separate heaters. Both the sample and reference are maintained at the same temperature and the difference in thermal power required to maintain them at the same temperature is measured and plotted as a function of temperature or time. The differential heat flow is therefore only due to the heat capacity associated with heating the sample. Small samples (5-10 mg) were weighed in an aluminium pan and the mass noted. The pan was then covered with its cover, usually slightly smaller. The pans are then crimped close using TA's Blue DSC sample press. The enclosed sample was placed in the furnace side by side with the crimped closed empty aluminium pan as reference. The instrument is purged with ultra pure N2 gas at regulated pressure between 100 and 140 kPa gauge (15 and 20 psig). The gas flow rate was set at 50 mL per min. Experiments were run from room temperature to 500 oC at scan rates of 10 oC/min.
3.5 Insulin-mimetic activity Insulin-mimetic studies were carried at the Diabetic Discovery Platform, South African Medical Research Council, Tygerberg, South Africa under the direction of Dr. Christo Muller.
3.5.1 In vitro studies 3.3.1.1 Cell culture Cells were cultured in Dulbecco modified Eagle’s medium (DMEM) with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and 4.5 g/L glucose (Lonza, USA) and 10 % fetal bovine serum (Highveld Biological, South Africa) in a humidified atmosphere of 5 % CO2 and 95% air at 37 oC. C2C12 cells were sub-
67
cultured in log phase to 70 % confluence and seeded at a density of 5000 cells/well into 96-well culture plates. To limit batch-to-batch variation cell subcultures were limited to ten passages. After three days culture myotube formation was induced by replacing the fetal bovine serum (FBS) in the medium with 10% horse serum (Highveld Biological, South Africa). All experiments were done in 5 days when more than 75% of the cells were differentiated morphologically.
3.5.1.2 Viable cell counts The cells were suspended in a trypan blue (0.1% w/w) phosphate buffered saline solution and the ratio of stained to non-stained cells was determined after 5 min of incubation time. Viable cell counts were performed by using a hemocytometer.
3.5.1.3 Glucose uptake determination Three hours prior to the glucose uptake, cells were incubated in glucose and serum free media. On the 5th day, the medium was removed and replaced with 50 μL modified DMEM without phenol red supplemented with 8 mM glucose and 0,1% BSA
(Sigma, USA) containing either the oxovanadium(IV) complexes at
concentration of 0.05 μg/μL or the positive controls, insulin or metformin at concentration of 1 μM was added to the 96-well plate. The plate was then incubated for 2 h at 37 oC and 5 % CO2. After incubation, 4 μL media was removed from each well and transferred to a new 96-well plate to which 196 μL deionized water was added in each well. 50 μL of this diluted medium was transfer to a new 96-well plate and 50 μL of the prepared glucose assay reagent (Biovision Inc., USA) was added per well and incubated for 30 min at 37 oC. Absorbance reading was measured at
68
wavelength 570 nm on a 96-well plate reader (Bio-Tek model ELx800, USA). The glucose concentration per well was calculated from a standard curve.
Glucose
utilisation was determined by subtracting the glucose concentration left in the medium of the relevant wells following incubation to media not exposed to cells during incubation. All assays were performed in triplicate.
3.5.1.4 Statistical analysis Statistical analysis of data was performed by means of the student’s t-test. The values are presented as means ± SD.
3.5.2 In vivo studies 3.5.2.1 Animals Adult male Wistar rats (400-450g), obtained from the Experimental Animal house of Diabetes Discovery Platform, South African Medical Research Council were bred in an air-conditioned room with controlled lighting 12:12 h light/dark cycle. Rats were allowed free access to standard solid food for laboratory animals and tap water. Diabetes was induced by a single intraperitoneal injection of STZ, at a dose of 36 mg /kg, to reduce or deplete the numbers of insulin producing cells and to induce hyperglycaemia at levels typical of type 1, or late stage T2D. Rats were fasted for 3 h but were provided with drinking water ad libitum. Blood samples were taken from the tail vein 72 h after STZ injection. Plasma glucose concentrations were determined with a glucometer (Accu-Check®, Roche Diagnostics, Mannheim, Germany). Rats with a blood glucose level of more than 300% of the fasting level were considered diabetic and were selected for the studies. Diabetic rats were divided into 10 experimental groups each containing five rats.
69
3.5.2.2 Experimental groups •
Control: given the solvent vehicle only i.e. water and DMOS 15% v/v, •
VOL1: was given 0.2 mM/kg of compound VOL1.
•
VOL2: was given 0.2 mM/kg of compound VOL2.
•
VOL3: was given 0.2 mM/kg of compound VOL3.
•
VOL4: was given 0.2 mM/kg of compound VOL4.
•
VOL5: was given 0.2 mM/kg of compound VOL5.
•
VOL6: was given 0.2 mM/kg of compound VOL6.
•
VOL7: was given 0.2 mM/kg of compound VOL7.
•
VOL12: was given 0.2 mM/kg of compound VOL12.
•
VOL13: was given 0.2 mM/kg of compound VOL13
The unsymmetrical Schiff base complexes of oxovanadium(IV) (VOL1-VOL4) were selected for exploratory in vivo analyses to establish the effect of the substituents observed under the in vitro analyses since the two cases of Schiff base complexes reported in literature for their insulin mimetics were on the symmetrical analogues [126-127]. Furthermore in vitro studies are expensive and highly controlled and the selection of this class of complexes is to cut down on cost.
3.5.2.3 Collecting of blood samples 0.2 mmol/kg solutions of the complexes in DMSO were administered orally to STZ-diabetic induced rats. Blood samples were taken from the tail vein and plasma glucose was measured at intervals of 0, 1, 2, 3, 4, 5 and 6 hours. 70
3.5.2.4. Analysis of data The percentage changes in the plasma glucose values of the treatment groups in the STZ rat model were calculated for each hour and then subtracted from the percentage changes in the control values (normalized against the control) at each time-point.
3.5.2.5 Gavage procedure Diabetic rats were lightly anaesthetized by inhalation of 98 % oxygen and 2 % fluothane
(AstraZeneca
Pharmaceuticals)
until
the
rats
were
sufficiently
anaesthetized to allow safe and stress-free handling while retaining their swallowreflex. A teflon gavage catheter was placed into the stomach, via the mouth and esophagus, and 1 mL of water and DMSO (15 % v/v) containing the VOL compound was injected directly into the stomach. An additional volume of approximately 200 μL of water was then injected to flush any remaining extract from the gavage catheter. The catheter was then promptly removed and the rat placed in its cage for recovery.
3.5.2.6 Statistical analysis Results were entered into an Excel spreadsheet and statistically analysed against the control at each time-point using the parametric t-test (Graphpad software).
A bar-graph with SEM error bars represents the hourly percentage
changes in the whole blood glucose values over the 6-h monitoring period.
A
statistical significance of p≤0.05 is indicated by *, statistical significance 0.01 – 0.0001 is indicated by ** and statistical significance p ≤ 0.0001 is normally indicated by ***.
71
CHAPTER 4 RESULTS AND DISCUSSION 4.1 Synthesis 4.1.1 Unsymmetrical Schiff bases A series of new structurally novel unsymmetrical Schiff base ligands were prepared by the condensation reaction in a 1:1:1 molar ratio of 2-hydroxy-1naphthaldehyde,
o-phenylenediamine,
and
substituted
salicylaldehyde
or
salicylaldehyde and kept stirring at room temperature for four days. All ligands formed were orange-yellow and melted at 134-195 oC. They were also obtained in high yield and in high purity (Table 4.1). The procedure for the preparation of the unsymmetrical Schiff bases was developed in our laboratory.
The sequence of
reaction is shown as part of the Scheme presented in Figure 4.1. The following factors were found to affect the course of the synthesis: (i) the sequence of addition of the reagents, (ii) temperature of the solution of the starting reagents (solution chilled to about 4 oC), (iii) the nature of the diamine (aliphatic or aromatic) used and (iv) the reaction time. A change of amine from o-phenylenediamine to 1, 3diaminopropane led to the formation of symmetrical Schiff base, N, N'-bis(2-hydroxy1-naphthalidene)-1, 3-diaminopropane, following the same procedure. From the above observation, it may be concluded that the resonance stabilization energy, arising from extended conjugation, reinforces the formation of the o-phen-bridged Schiff base as against the symmetrical Schiff base formed with the aliphatic diamine. A change in reaction time from four days to just three hours or the use of warm solutions of the starting reagents produced mixed products. From the above observations, it seems that the condensation reactions occurred stepwise.
72
O C
H
O
NH2
C
H
NH2
OH
+
OH
+
R
EtOH
H
H C
N
N
C R
OH
R
HO
H2L1 H 2L
M(CH3COO)2 VOSO4.xH2O
H2
MeOH
or
L3
H 2L4
Et3N
Cl NO 2 OCH 2CH3 H
O
O
MII C
2
R N
N
C H
H
M = VO 2+, Co2+, Ni2+,Cu2+
Fig. 4.1 Preparation of the unsymmetrical Schiff bases and their metal(II) complexes 4.1.2 Unsymmetrical Schiff base metal(II) complexes A series of new structurally novel unsymmetrical tetradentate Schiff base metal(II) complexes of oxovanadium(IV), cobalt(II), nickel(II), and copper(II) were prepared by refluxing together the relevant unsymmetrical Schiff bases with the corresponding oxovanadium(IV) sulphate (or metal(II) acetate for other metals) in 73
methanolic medium and is shown as part of the Scheme presented in Figure 4.1. They were isolated pure from methanol in very good yields. The purity of the metal complexes was established by microanalyses as formulated. All the unsymmetrical Schiff base complexes did not melt or decompose when heated up to 250 oC. The analytical data, colour, percentage yields, and melting points of the complexes are presented in Table 4.1.1− 4.1.2.
4.1.2.1 Unsymmetrical Schiff base complexes of cobalt(II) The
cobalt(II)
complexes
were
prepared
by
refluxing
the
relevant
unsymmetrical Schiff bases with Co(CH3COO)2·4H2O in methanol. The resulting brown solid are stable in air.
4.1.2.2 Unsymmetrical Schiff base complexes of nickel(II) The nickel(II) complexes were synthesized by reacting the respective unsymmetrical Schiff bases with Ni(CH3COO)2·4H2O in 1 : 1 molar ratio in methanol. All the nickel(II) complexes formed were red in colour.
4.1.2.3 Unsymmetrical Schiff base complexes of copper(II) The copper(II) complexes were prepared by refluxing appropriate amount of Cu(CH3COO)2·H2O and the respective unsymmetrical Schiff bases in methanol. All the complexes were pale brown in colour.
4.1.2.4 Unsymmetrical Schiff base complexes of oxovanadium(IV) The oxovanadium(IV) complexes were prepared by heating a mixture of each ligand and oxovanadium(IV) sulphate in 1:1 metal:ligand ratio, buffered with
74
triethylamine at 50 oC. All the oxovanadium(IV) complexes formed were green in colour as shown in Table 4.1.1. 4.1.3 Symmetrical Schiff bases and their complexes The symmetrical ligands and their oxovanadium(IV) complexes were isolated pure from ethanol and methanol respectively, and in very good yields. All the ligands involving aliphatic bridges are yellow while those involving aromatic diamine are either orange or orange-yellow in colour. All the trimethylene-bridged complexes are orange. VOL10 and VOL17 are brown and others are green in colour.
All the
complexes did not melt/decompose up to 250 oC except VOL19 which melted at 239240 oC. The high melting points of these complexes reflect some molecular complexity. Complexes VOL7 and VOL17 were formed with associated MeOH to form distorted octahedral complexes. The analytical data, colour, percentage yields, and melting points of the ligands and complexes are contained in Table 4.1.1
4.1.3.1 Benzophenoneimine The Schiff bases were prepared by refluxing equimolar (0.08 mol) quantities of 2-hydroxybenzophonone in 60 mL absolute ethanol and the respective aliphatic diamine (0.04 mol) in ethanol (10 mL) for 2 h. All the ligands were yellow and melted at 123-165 oC. The oxovanadium(IV) complexes were isolated from the reaction of VOSO4 with the preformed Schiff bases in methanol in 1:1 metal to ligand ratio, buffered with triethylamine. They were all green except the complex involving the trimethylene bridge, which gave an orange colour and was also formed with associated MeOH
75
4.1.3.2 Naphthaldiimines The Schiff bases were prepared by heating equimolar (0.08 mol) quantities of 2-hydroxy-1-naphthylaldehyde in 60 mL absolute ethanol and the respective aliphatic/aromatic diamine (0.04 mol) in ethanol (10 mL) under reflux for 2 h. All the Schiff bases in this series were yellow except the one with the aromatic amine bridge which gave an orange product. All the Schiff bases melted at 222-234 oC except H2naph2-en which melted over 250 oC. Reactions of the Schiff bases with VOSO4 in methanol in 1:1 metal to ligand ratio, buffered with triethylamine, gave green powders of VOL8, orange powders of VOL9 and brown powders of VOL10.
4.1.3.3 Chlorosalicylaldiimines The Schiff bases were prepared by condensation reaction of 5-chloro salicylaldehyde (0.08 mol) with the respective diamine (0.04 mol) in absolute ethanol for 2 h and recrystallized from ethanol-chloroform (1:3 v/v) mixture. All the ligands were yellow except H2Clsal2opd which was orange-yellow in colour. All the Schiff bases melted at 61- 220 oC. The oxovanadium(IV) complexes were isolated from the reaction of VOSO4 with the preformed Schiff bases in methanol and buffered with triethylamine. Complexes VOL11 and VOL12 are green, and VOL14 and VOL13 are yellow and orange-yellow respectively.
4.1.3.4 Methoxysalicylaldiimine The Schiff bases were prepared by heating equimolar (0.08 mol) quantities of 5-methoxysalicylaldehyde
in
60
mL
absolute
ethanol
and
the
respective
aliphatic/aromatic diamine (0.04 mol) in ethanol (10 mL) under reflux for 2 h. The ligands in this series were all yellow except H2Omesal2opd which gave an orange
76
product. All the Schiff bases melted at 82-166 oC. The oxovanadium(IV) complexes of the series were formed by the reactions of the Schiff bases with VOSO4 in methanol and was buffered with triethylamine. The products gave green powders for VOL15, orange powders for VOL16 and brown powders for VOL17.
4.1.3.5 Ethoxysalicylaldiimine The Schiff bases in this series were prepared by refluxing equimolar (0.08 mol) quantities of 3-ethoxysalicylaldehyde in 60 mL absolute ethanol and the respective aliphatic/aromatic diamine (0.04 mol) in ethanol (10 mL) for 2 h. All the ligands were yellow except H2Oetsal2opd, which gave an orange product and their melting points were in the range of 70-139 oC. The oxovanadium(IV) complexes were isolated from the reaction of VOSO4 with the preformed Schiff bases in methanol, buffered with triethylamine. All the complexes in this series were green and they did not melt/decompose below 250 oC, except the methylethylenediaminebridged complex which melt at 239-240 oC.
4.2 1H NMR spectra of the Schiff bases The 1H NMR spectral data of the free ligands recorded in CDCl3 against tetramethylsilane (TMS) as internal reference are presented in Table 4.2. The 1HNMR spectra of the Schiff bases displayed the O―H protons of the phenolic groups and azomethine protons (H—C=N) at 12.7-15.3 and 8.3-9.5 ppm, as singlets, respectively. The aromatic protons, which appeared as a multiplet, were observed at 6.6-8.1 ppm. The signal due to the methyl protons were observed at 1.3-3.8 ppm while the CH2 protons were observed between 2.0 and 4.1 ppm. The C—H protons present only in methylethylenediamine-bridged ligands were observed at 3.5-3.8
77
ppm. The chemical shifts obtained were similar to those of Schiff bases reported in the literature [60, 61, 176, 177].
4.2.1 Unsymmetrical Schiff bases The appearance of two different peaks for each of the azomethine protons and phenolic protons confirm the unsymmetrical nature of the Schiff bases in this series. The higher of the two signals for both the azomethine and phenolic protons is assigned to the azomethine/phenolic proton attached to the naphthaldehyde ring while the lower signals is assigned to the azomethine/phenolic proton attached to salicylaldehyde ring. Signals for the methine proton of the azomethine group were observed between 8.2 and 9.0 ppm. The peaks in the region 6.8-8.1 ppm, which appeared as a multiplet, are assigned to chemical shifts for aromatic protons. The O―H protons of the phenolic group were observed as a singlet between 12.0 and 15.5 ppm and were generally shifted downfield due to intramolecular hydrogen bonding [60]. The signal due to the methyl protons on the ethoxy substituent in H2L3 appeared as a triplet at 1.5 ppm while the signal at 4.1 ppm is assigned to the CH2 proton.
4.2.2 Symmetrical Schiff bases The 1H NMR spectral of the symmetrical Schiff bases showed single peaks for each of the azomethine protons and phenolic protons. This is attributed to the symmetrical nature of these ligands.
78
4.2.2.1 Benzophenoneimine The signal corresponding to the azomethine protons were absent in this series because the hydrogen atom is replaced by the phenyl group. 1H NMR spectra gave the aromatic protons in the range of 6.6-7.5 ppm and the O―H protons of the phenolic groups in the range of 15.2-15.5. Signals for the methyl protons were observed at 1.3 ppm while the CH2 protons were observed between 2.1 and 3.7 ppm. The CH proton of the methylethylenediamine ligand was observed at 3.5 ppm.
4.2.2.2 Naphthaldiimines The O―H protons of the phenolic groups and the azomethine protons of this series appeared at 14.3-15.1 and 8.2-9.5 ppm respectively, as a singlet. The peaks for the aromatic protons were observed at 7.0-8.1 ppm as a multiplet. The CH2 protons of the trimethylenediamine bridged ligand appeared as two different peaks corresponding to two different proton environments. The middle CH2 protons appeared as a multiplet at 2.2 ppm and others appeared as a triplet at 3.8 ppm. The CH2 protons for the ethylenediamine bridged ligand were observed at 4.0 ppm as a singlet.
4.2.2.3 Chlorosalicylaldiimines The 1H NMR spectral of this series showed the aromatic protons as a multiplet in the range 6.6-7.4 ppm and the O―H protons of the phenolic groups in the range 13.0-13.4 ppm. The azomethine protons appeared as a strong singlet in the range 8.3-8.6 ppm. The peaks for the methyl protons were observed at 1.3 ppm while the CH2 protons were observed between 2.1 and 4.0 ppm. The CH proton of the methylethylenediamine bridged ligand was observed at 3.7 ppm.
79
4.1.2.4 Methoxysalicylaldiimine In the 1H NMR spectral of this series, the signals due to the azomethine protons appeared as a singlet at 8.3-8.6 ppm while the O―H protons of the phenolic groups were observed at 12.7-12.9 ppm. The signal due to the methoxy protons was observed as a singlet at 3.8 ppm. Three peaks were observed for the CH2 protons at 2.2, 3.7 and 4.0 ppm, corresponding to a multiplet, a triplet and a singlet respectively.
4.1.2.5 Ethoxysalicylaldiimine The 1H-NMR spectra of the Schiff bases in this series exhibit a multiplet signals at 6.7–7.3 ppm which are attributed to the aromatic protons. The methyl protons of the ethoxy substituent were observed as a triplet at 1.5 ppm while its CH2 protons appeared as a quadruplet at 4.1-4.2 ppm. The methyl protons of the methylethylenediamine bridged ligand were observed as a duplet at 1.4 ppm while its CH2 protons were observed as a doublet at 3.9 ppm. The signals due the azomethine protons and the O―H protons of the phenolic groups were observed as a singlet at 8.3-8.6 and 13.1-13.8 ppm respectively
.
For the unsymmetrical Schiff bases, the observed difference in the signal of
the phenolic proton is attributed to the greater electron withdrawing effect of the naphthaldehyde ring and hydrogen bonding between the phenolic proton and the azomethine nitrogen. The deshielding effect of electron withdrawing groups and hydrogen bonding are responsible for the proton signal being moved further downfield. Similarly, the same trend is observed for the azomethine proton of the
80
unsymmetrical Schiff bases. The difference observed in the methyl proton signals of the methoxy and the ethoxy groups is attributed to the closeness of the protons from the deshielding effect of the electron withdrawing oxygen atom in the group while the methyl proton of the methylethylenediamine bridged is observed upfield because it is well shielded. The shift in the phenolic proton of the benzophenoneimines downfield can be accounted for by either the presence of hydrogen bonding between the phenolic proton and the azomethine nitrogen or tautomerism in the 2-hydroxy substituted imines.
4.3 Infrared spectra The important infrared spectral bands of the ligands and the metal(II) complexes are presented in Table 4.3.1−4.3.2 and Fig. 4.3.1-4.3.6. The tentative assignments of the observed bands for the compounds were made by comparing the spectra with those reported in the literature on similar systems [40, 41, 59]. The absorption bands due to the amino group disappeared in the IR spectra of all the ligands, which showed that the amino groups in the diamine condensed with the aldehyde/ketone. The band appearing at 1567-1640 cm– I due to azomethine group in the ligands is shifted to lower frequency at 1573-1634 cm–I in the metal(II) complexes, indicating the participation of the azomethine nitrogen in interaction with the metal ion. Similarly, the ν(C―O) band of the ligands, which occurs at 1257-1334 cm–1, shifted to 1277-1366 cm–1 in the complexes indicated deprotonation and coordination of the phenolic oxygen to the metal atom. Thus, it can be concluded that the Schiff bases acted as tetradentate ligands coordination via the azomethine N and the phenolic O. Further conclusive evidence of the coordination of the Schiffbases with the metal ions was shown by the appearance new bands at 435-583 and 81
410-581 cm−l assigned to the metal nitrogen (M―N) and metal-oxygen (M―O) vibrations, respectively. These bands were absent in the spectra of the uncomplexed Schiff bases, thus confirming participation of the O and N atoms in the coordination. It has been established that the metal-ligand vibrational modes are very sensitive to substituent effects [178, 179]. This was proposed on the basis of isotopic labelling studies (15N- and
18
O-labelling). The substituent effects were based on the position
of substitution rather than on the nature of the substituents. The ν(M―O) bands are observed to exhibit higher vibrational frequencies than the ν(M―N) bands for the meta-substituents while the order is reversed for the para-substituents regardless of the nature of the substituents.
It was suggested that the transmission of the
substituent effects in the Schiff base complexes are propagated largerly by a mesomeric mechanism. The important bands along with their assignments are listed in Table 4.3.1.-4.3.2 and representative spectra are shown in Fig. 4.3.1-4.3.6. Oxovanadium(IV) complexes of Schiff bases can give rise either to monomeric
structures
with
square
pyramidal/trigonal
bipyramidal/octahedral
coordination geometry or to polymeric structures, involving ····O=V····V=O···· linkages, with a distorted octahedral geometry [40]. However, on the basis of the location of the ν(V=O) band it is possible to distinguish between monomeric and polymeric complexes. In the present work, most of the oxovanadium(IV) complexes exhibit a strong band in the region 959-988 cm−1, which have been assigned to ν(V=O) with a monomeric square pyramidal coordination geometry. All the orangeyellow coloured complexes, with the trimethylene bridge in which the ν(V=O) appeared at 848-860 cm–1, have been assigned polymeric species with ····O=V····V=O···· interactions, which afforded a distorted octahedral coordination geometry. 82
4.3.1 Unsymmetrical Schiff bases and their complexes The infrared spectra of both the ligands and complexes have no bands between 3100 and 4000 cm–1, indicating the absence of the uncondensed N―H and uncoordinated —OH groups. Due to the unsymmetrical nature of the ligangs and the complexes, two bands were observed for each of the following bonds: ν(C=N), ν(C―O), ν(V―N) and ν(V―O), taking their origin from the different aldehydes.
4.3.1.1 Unsymmetrical Schiff base complexes of cobalt(II) The position of ν(C=N) bands of the ligands appeared at 1610−1621 and 1567−1583 cm–1. These are shifted to lower frequencies at 1603−1606 and 1571−1578 cm–1 respectively upon complexation indicating the involvement of the nitrogen atom of the azomethine group in coordination [176]. On the other hand, the (C―O) stretching frequencies, which occur at 1313−1333 and 1276−1289 cm–1 for the ligands was moved to higher frequencies by 12−30 cm–1 after complexation, which indicates that the shifts are due to coordination of the phenolic oxygen of the ligand to the metal ion [175]. The new bands observed in the complexes in the region 453−580 and 508−554 cm–1 were assigned to ν(Co―N) while 463−575 and 423−428 cm–1 are attributed to ν(Co―O) [176, 179].
4.3.1.2 Unsymmetrical Schiff base complexes of nickel(II) The spectra of the ligands show two different C=N stretching frequency at 1610−1621 and 1567−1583 cm–1, which are shifted to lower frequencies in the spectra of all the nickel complexes at 1603−1609 and 1577−1582 cm–1 respectively indicating the involvement of azomethine nitrogen in coordination to the metal ion 83
[176]. The corresponding phenolic C―O stretching frequency occurs at 1313−1333 and 1276−1289 cm–1 for the ligands and at 1331−1366 cm–1 and 1287−1311 cm-1 for the complexes. The shift to a higher frequency of this band confirms the participation of the phenolic oxygen in bonding [177]. Assignment of the proposed coordination sites is further supported by the appearance of new bands at 457−583 and 507−554 cm–1 which are assigned to ν(Ni―N) while 463−577 and 431 cm–1 are attributed to ν(Ni―O) [176, 179].
4.3.1.3 Unsymmetrical Schiff base complexes of copper(II) The ligands exhibited the characteristic C=N stretching frequency at 1610−1621 and 1567−1583 cm–1. The shifting of ν(C=N) band to lower values by 10−12 cm–1 indicates the participation of the two azomethine nitrogen atoms in bonding [176]. The corresponding phenolic C―O stretching frequency occurs at 1313−1333 and 1276−1289 cm–1 for the ligands and at 1325−1363 cm–1 and 1290−1317 cm–1 for the complexes. The shift in C―O stretching frequency confirms the participation of the phenolic O in C―O―M bond formation [175]. The bands due to ν(Cu―N) observed only in the complexes occurred at 460−574 and 501−548 cm–1 while 460−574 cm–1 and 416−463 are attributable to ν(Cu―O) bond [176, 179]. These bonds were observed in the spectra of the metal complexes and not in the spectra of the Schiff bases.
4.3.1.4 Unsymmetrical Schiff base complexes of oxovanadium(IV) The ligands exhibited the characteristic C=N stretching frequency at 16101621 and 1567-1583 cm–1. These are shifted to lower frequencies at 1604-1607 and 1573-1576 cm–1 respectively upon complexation indicating the involvement of the 84
nitrogen atom of the azomethine group in coordination [176]. The corresponding phenolic C―O stretching frequency occurs at 1313-1333 and 1276-1289 cm–1 for the ligands and at 1361-1366 cm–1 and 1312-13424 cm-1 for the complexes. The shift in C―O stretching frequency confirms the participation of the phenolic O
in
C―O―M bond formation [175]. A significant change observed in the infrared spectra of the complexes is the appearance of strong absorption band due to νV=O, which is absent in the ligands. The frequency spread observed for a large number of oxovanadium(IV) complexes was put at 985±50 cm–1 [165]. All the complexes exhibited a strong V=O stretching band at 970-988 cm–1, which confirms that the complexes are monomeric [158]. The bands observed in the complexes in the region 483-558 and 536-541 cm–1 were assigned to ν(V―N) while 488-581 and 455-458 cm-1 are attributed to ν(V―O) [176]. Provisionally, the lower frequency bands of the azomethine group may be assigned to the νC=Nnaph and the higher frequency bands to the νC=Nsal of the unsymmetrical Schiff bases.
4.3.2 Symmetrical Schiff bases and their complexes 4.3.2.1 Benzophenoneimine All the Schiff bases reported gave a sharp and strong band due to ν(C=N) of the azomethine group at 1604-1607 cm–1 while the corresponding bands in the complexes were observed in the 1599-1601 cm–1 range. The observed shift to a lower frequency of the ν(C=N) in the complexes indicates a decrease in the bond order of C=N due to the coordination of the azomethine nitrogen to the metal ion [29]. Similarly, the ν(C―O) band of the ligands, which occurs at 1331-1334 cm–1, 85
shifted to 1336-1337 cm–1 in the complexes which suggests the coordination of the phenolic oxygen with the metal ion. All the oxovanadium(IV) complexes in this series exhibit a strong band in the range 959-986 cm−1, which has been assigned to ν(V=O) in a monomeric square pyramidal coordination geometry. The complex [VO(bp2-tn)MeOH], behaves differently as the ν(V=O) occurs at 959 cm−1 confirming that the MeOH behaves as an adduct and excludes polymerization. The bands due to ν(V―N) and ν(V―O), observed only in the complexes, occurred at 495-528 and 406-489 cm–1 respectively [41].
4.3.2.2 Naphthaldiimines IR spectra of these Schiff bases were also compared with the spectra of the metal complexes. The bands at 1615–1634 cm–1 due to the azomethine group of the Schiff base underwent a shift to lower frequency (1600–1618 cm–1) after complexation, indicating the coordination of azomethine nitrogen to metal atom. The ν(C―O) band of the ligands, which occurs at 1257-1287 cm–1, shifted to 1281-1343 cm–1 in the complexes confirming coordination of the phenolic oxygen with the metal ion. VOL8 and VOL10 exhibit a strong band in the range 979-988 cm−1, which have been assigned to ν(V=O) in a monomeric square pyramidal coordination geometry. The orange coloured complex, with a trimethylene bridge in which the ν(V=O) appeared
at
852
cm–1,
has
been
assigned
a
polymeric
structure
with
····O=V····V=O···· interactions, which afforded a distorted octahedral coordination geometry [40]. New bands, absent in the spectra of Schiff bases, appeared at 549-577 and 466-506 cm–1 and are attributed to ν(V―-N) and ν(V―O) vibrations, respectively. 86
The appearance of ν(V―N) and ν(V―O) vibrations supports the involvement of nitrogen and oxygen atoms in complexation [41, 176].
4.3.2.3 Chlorosalicylaldiimines The band at 1615-1636 cm−1 in the ligands, which shifts to 1612-1630 cm−1 in the complexes, is assigned to ν(C=N) frequency. The ν(C=N) frequency is displaced to lower frequency, indicating a decrease in the C=N bond order due to the coordinate bond of the metal with the lone pair of the azomethine nitrogen. The ν(C―O) band of the ligands, which occurs at 1277-1280 cm–1, was slightly displaced to higher frequencies (1297-1322 cm–1) on coordinated to the metal complexes. The characteristic ν(V=O) stretching frequency in the oxovanadium(IV) complex appears as a strong band at 960-988 cm–1, within the range 960±50 cm–1 reported for square pyramidal oxovanadium(IV) complexes [165]. The ν(V=O) stretching frequency of the trimethylene derivative occurs at 860 cm–1, which is consistent with polymeric system involving ····O=V····V=O···· bridge. Assignment of the proposed coordination sites is further supported by the appearance of new bands at 516-554 and 435-498 cm–1 which are attributed to ν(V―O) and ν(V―N) respectively [41]. These bands are observed as new absorption peaks of the complex that are not present in the spectra of the free ligand.
4.3.2.4 Methoxysalicylaldiimines The ligands exhibited the characteristic C=N stretching frequency at 16221640 cm−1 and are shifted to lower frequencies (1599-1634 cm-1) upon complexation indicating the involvement of the nitrogen atom of the azomethine group in 87
coordination [59]. The phenolic C―O stretching frequency occurs in the 1272-1285 cm−1 for the ligands and in the 1277-1307 cm−1 region for the complexes. The C―O stretching frequency is generally shifted to a higher frequency, indicating the participation of phenolic oxygen in C―O―M bond formation [175]. A significant change observed in the infrared spectra of the complexes compared to their respective ligands is the appearance of strong absorption band due to V=O stretching frequency in the complexes. All the complexes exhibited a strong V=O stretching band at 979-981 cm-1, which suggests monomeric square pyramidal structures. The ν(V=O) stretching frequency of the trimethylene derivative occurs at 848 cm–1, which is also consistent with polymeric system involving ····O=V····V=O···· bridge. Other bands observed in the complexes in the regions 544-573 and 476-497 cm-1 are due to ν(V―O) and ν(V―N) respectively [41]. These bands were absent in the spectra of the ligands.
4.3.2.5 Ethoxysalicylaldiimines The spectra of the Schiff bases show ν(C=N) bands in the region 1614-1637 cm–1, which is shifted to lower frequencies in the spectra of all the complexes (16021631 cm–1) indicating the involvement of nitrogen in coordination to the metal ion. The ν(C―O) band of the ligands, which occurs at 1272-1283 cm–1, was slightly displaced to higher frequencies (1304-1315 cm–1) on coordinated to the metal complexes. The characteristic ν(V=O) stretching frequency in the oxovanadium(IV) complex appears as a strong band at 960-988 cm–1, which is consistent with monomeric square pyramidal structures. The nature of metal–ligand bonding is confirmed by the newly formed band at 541-614 and 448-483 cm–1 in the spectra of
88
the complexes which are tentatively assigned to ν(V―O) and ν(V―N) respectively [41].
4.4 Electronic spectra of the metal(II) complexes The electronic absorption spectra are often very helpful in the evaluation of results furnished by other methods of structural investigation. The electronic spectral measurements were used for assigning the stereochemistries of metal complexes based on the positions and number of d–d transition peaks. The electronic absorption spectra of metal(II) complexes were recorded in 10−3 M and 10−5 M solutions of each complex in DMSO and chloroform in the range 200−1100 nm at room temperature. The electronic absorption spectra of cobalt(II), nickel(II) and copper(II) in 10−3 M solutions did not form homogeneous solutions and the spectra were noisy in both the visible and the UV regions. The results of the solution spectra are presented in Figure 4.4.1−4.4.4 and Table 4.4.1−4.4.2. For four-coordinate cobalt(II), nickel(II) and copper(II) complexes, either a square planar or a tetrahedral configuration is possible. On the basis of electronic spectra, together with the magnetic moments, it is possible to differentiate between these two configurations. The optical spectra of square pyramidal oxovanadium(IV) complexes are characterized by three d-d transitions. These transitions have been assigned using the energy level scheme proposed by Ballhausen and Gray for C4v symmetry [87]. In this scheme band I is assigned as b2→ eπ* (11 000–16 000), band II as b2→b1* (14 500–19 000) and band III as b2→ a1* (21 000–30 000). As mentioned earlier, Selbin[165] has questioned the general applicability of the Ballhausen and Gray scheme and has suggested that the ground level is the singly occupied, nonbonding 89
b2 orbital but that the eπ* and b1* levels may be very close in energy such that they may cross one another from complex to complex. provide experimental evidence for
the inversion of
Kolawole, et al. [40, 41] did eπ* and b1* in a series of
symmetric Schiff base complexes of oxovanadium(IV) as the methylene bridge increases from 2 to 10 and proposed an energy level which incorporates Ballhaussen and Gray, and Selbin’s schemes. An overlap between band III and charge transfer has always been a problem in being able to make a definitive assignment of band III.
It tends to ‘borrow’
intensity from a nearby charge transfer transition, which in many instances reduces it to a shoulder.
It has been possible to assign the three bands in most of the
complexes reported in this work, except that band III, expectedly, appears as a shoulder in some of the complexes. In the complexes where the intensity of band I is lower than the intensity of band II inversion in the energies levels of b1* and eπ* has been invoked. Only bands II and III could be observed in the solutions spectra of the unsymmetrical Schiff base complexes. Complexes of oxovanadium(IV) with coordination numbers 5 and 6 are usually square pyramidal/trigonal bipyramidal and distorted octahedral, respectively. Due to a strong axial field, the energy levels associated with these structures do not differ considerably and the same scheme has been used for the interpretation of the spectra of oxovanadium(IV) complexes. In five-coordinate complex band I is simply shifted towards lower wavelengths and only the complexes with C2v symmetry display four absorption bands between 400 and 860 nm. A reduction in the molar absorptivity of band II in spectra run in a coordinating solvent or its disappearance could be indicative of some sixth-coordination in such solvent.
90
4.4.1 Unsymmetrical Schiff base metal(II) complexes 4.4.1.1 Unsymmetrical Schiff base complexes of cobalt(II) The electronic spectra of all the cobalt(II) complexes in CHCl3 are very similar to each other and consist of three bands, one each at 18 587−18 939, 22 272−22 779 and 24 876–25 575 cm−1 regions, which clearly indicate the low-spin square planar/distorted square planar geometry of the complexes [180]. This is also corroborated by the observed effective magnetic moment of the complexes. The other intense bands between 28 902 and 41 494 cm−1 are due to charge transfer transitions. The electronic spectra of all the complexes in DMSO displayed a single d-d transition at 25 063−25 445 cm−1 and a charge transfer transition at 29 412−36 101 cm−1. The appearance of a single d−d transition in DMSO is attributed to the effect of the coordination of solvent which alters the stereochemistry to form low spin six-coordinate distorted octahedral [181]. The slight differences in the peaks observed in the spectra of these complexes in both solvents are due to substituent effects of the different substituents on the salicylaldehyde ring
4.4.1.2 Unsymmetrical Schiff base complexes of nickel(II) The solution spectra of all the nickel(II) complexes in both solvents are very similar to each other and consist of four bands. The electronic spectra of the complexes in CHCl3 show two bands at 20 619−20 747 and 25 773−26 178 cm−1 assignable to 1A1g→1B1g and 1A1g→1Eg transitions in a square-planar geometry [177]. The assignment of square-planar geometry is supported by the zero BM effective magnetic moment the complexes. The stereochemistry of these complexes was unchanged when moving from a non-coordinating solvent to a coordinating solvent (DMSO) except for the complex [NiL2], whose ligand contains the nitro group, which
91
shows a slightly deviation from the other. The observed bands in this complex were still in conformity with a squre-planar geometry. The solution spectra of the complexes in DMSO also show two bands at 20 619−20 877 and 25 974−26 178 cm−1. At higher energy, two more strong absorptions are observed in the range 30 303−38 760 (CHCl3) and 30 395−39 063 cm−1 (DMSO) which are likely due to charge transfer or intra-ligand transitions.
4.4.1.3 Unsymmetrical Schiff base complexes of copper(II) The electronic spectra of all the copper(II) complexes in both CHCl3 and DMSO show a broad unsymmetrical band centred at 22 831−24 038 cm−1 and 23 095−24 038 cm−1 respectively. This broad band is assigned to 2B1g→2A1g transition of four-coordinate square-planar geometry [60, 177]. This d-d transition is in the region of that observed for structurally well-characterized complexes of coper(II) Nalkylsalicyladiminates with square-planar geometry [60]. The other intense bands between 27 778 and 40 816 cm−1 in CHCl3 and 28 490−37 313 cm−1 in DMSO are due to charge transfer or intra-ligand transitions
4.4.1.4 Unsymmetrical Schiff base complexes of oxovanadium(IV) Three structural types can be identified from the solution spectra of this series. VOL1 and VOL4 have similar spectra in both solvents. VOL2 and VOL3 are distinctively different in chloroform and DMSO. The electronic spectra in both solvents for 10–3 M solution displayed a broad d-d transition similar to the one observed by Kolawole, et al. [182] for symmetric napthaldiimine complexes. The authors also proposed the possibility of pseudoaromatization of the rings around V when the azomethine N atoms are bridged with aromatic groups. In such instances
92
the d1 electron of the V could be delocalised into the ring system. Similar scenario is suspected in these complexes because the salicylidiimine group is also bridged with a phenyl ring in each case. Such a conjugation would cause ligand based electronic transition to shift to red overlapping with the d-d transition resulting in complex spectra, like the one recorded here in solution. At 10–3 and 10–5 M solutions only bands II and III could be observed in these complexes. The high extinction co-efficient observed for band III corroborate the borrowing of intensities from the ligand (as a result of the hyperconjugation) by the vanadium. The d-d bands are assigned as follows: b2→b1*: 16 207–16 393 (CHCl3), 15 748–16 129 (DMSO) and b2→ a1*: 21 598–24 096 (CHCl3), 23 474–24 096 (DMSO). The bands in the region 27 701–41 322 (CHCl3) and 27 473–38 610 (DMSO) are assigned to either a metal-ligand charge transfer band or to an electronic transition within the ligand. The observed shift in band II of this series with a lowering of intensity in the molar absorptivity from CHCl3 to DMSO is indicative of solvent interaction in DMSO while they remain five-coordinate in CHCl3. The above observation may indicate a facile formation of six-coordinate species in DMSO.
4.4.2 Symmetrical Schiff base complexes of oxovanadium(IV) 4.4.2.1 Benzophenoneimino complexes The spectra of the chloroform solutions of VOL5 and VOL6 show two d-d transition in the visible region, the first at 16 026–17 182 cm−1 and the other at 20 747–21 277 cm−1 (often as a shoulder). The spectrum in DMSO of VOL5 is characterized by two bands at 13 870–17 575 cm−1 and a shoulder at 21 277 cm−1 while the spectrum of VOL6 gave only one band at 17 123 cm−1 and a shoulder at 21 276 cm−1. The spectra of the solutions in DMSO of these two complexes were
93
notably different. The two bands observed in chloroform solutions were shifted to 17 123-19 231 cm−1 and 21 097-21 277 cm−1 respectively, in DMSO. Complex VOL3 displayed the three spectral peaks predicted for VO2+ complexes and appears to assume octahedral geometry in DMSO. Most trimethylene-bridged Schiff base complexes reported were, like this compound, orange, but more planar, polymeric, (with ····V=O····V=O···· linkages) and distorted octahedral [182]. The only evidence that supports the planarity of this compound is that it is yellow and it was formed with associated methanol molecule, which reduces the νV=O to 959 cm–1 as evidence of the solvent perturbation of the V=O bond (unsolvated νV=O for the other complexes occur at about 990 cm–1). The change of solvent from chloroform to DMSO resulted in some shifts in band positions and resulted in enhanced intensity in some of the d-d and CT transitions. The bands are assigned as follows: b2→b1*: 12 555–13 263 (CHCl3), 12 555–12 970 (DMSO); b2→eπ*: 17 182–17 544 (CHCl3), 17 123–19 231 (DMSO) and b2→a1*: 20 747-21 186 (CHCl3), 21 097–21 277 (DMSO). The bands in the region 27 027–41 322 cm−1 are assigned to charge-transfer or intra-ligand transitions. The above spectral characteristics indicate that VOL5 and VOL6 exhibit five-coordinate geometry in non-coordinating solvent, CHCl3, whereas they exhibit six coordination number in coordinating solvent, DMSO, in which the sixth coordination site is occupied by the solvent molecule. VOL7 displayed six-coordinate geometry in both non-coordinating and coordinating solvents.
4.4.2.2 Naphthaldiimino complexes Two characteristic bands out of the three predicted for oxovanadium(IV) complexes in the visible region were displayed in the solution spectra of this series 94
except complex VOL9 which exhibits only band III in CHCl3. A considerable reduction in molar absorptivities was observed on going from CHCl3 to DMSO in the solution spectra of VOL8 and VOL10. These complexes are five-coordinate square pyramidal in CHCl3 and the reduction in molar absorptivity in DMSO indicates a facile formation of six-coordinate species in this solvent. Distinct spectral change was observed in the solution spectrum of VOL9 when this complex was treated with DMSO. Band III disappears and two new bands appear at 12 887 and 19 455 cm−1 which are assigned as band I and band II respectively. Band II transition has a greater intensity than band I in this complex, indicative of inversion of the eπ* and b1* energy-levels [40]. This complex appears to be six-coordinate in DMSO and like most derivatives of trimethylene-brigded complexes, it is polymeric (with ····V=O····V=O···· linkages) and distorted octahedral geometry in the solid state. The bands are assigned as follows: b2→b1*: 12 887 (DMSO); b2→eπ*: 15 942–16 155 (CHCl3), 15 528–19 455 (DMSO) and b2→a1*: 21 749–25 840 (CHCl3), 21 786–23 641 (DMSO). In the UV region, some chargetransfer or intra-ligand bands are observed between 26 178 and 40 650 cm−1.
4.4.2.3 Chlorosalicylaldiimino complexes Complex VOL12 displayed the three spectral peaks predicted for VO2+ complexes in DMSO and two bands in CHCl3. The shift and reduction in molar absorptivities in the band II of complex VOL12 observed on going from CHCl3 to DMSO is indicative of different stereochemistry in both solvents. It appears to exhibit five-coordinate geometry in CHCl3 whereas, it exhibits six coordination in DMSO in which the sixth coordination site is occupied by the solvent molecule. Band I and III were absent in the solution spectra of VOL11 and the band II observed was shifted to 95
higher energy with a reduction in the molar absorptivities. The similarity in the band II in the spectra of VOL12 and VOL11 might be as a result of both possessing similar structures in these solvents. Complex VOL13 was not soluble in both solvents and complex VOL14 was only sparingly soluble in both solvents. The spectrum VOL14 was noisy in the visible region run on 10-3 M solution and some of the bands in the region could not be extracted. Only band III was observed in the spectrum of VOL14. The bands are assigned as follows: b2→b1*: 13 263 (CHCl3), 12 970 (DMSO); b2→eπ*: 16 778–16 892 (CHCl3), 17 544–17 699 (DMSO) and b2→a1*: 21 186 (DMSO). In the UV region, some charge-transfer or intra-ligand bands are observed at 26 178–41 152 cm-1.
4.4.2.4 Methoxysalicylaldiimino complexes The spectra solutions of VOL15 in both solvents show only two d-d transitions out of the three predicted for oxovanadium(IV) complexes. Band I transition was absent in the solution spectra of VOL15 in both solvents and band II experiences a shift of ~197 cm−1 with a lowering of intensity in the molar absorptivity from CHCl3 to DMSO which is indicative of six-coordination geometry in DMSO solution but five coordinate geometry in CHCl3. Complex VOL16 formed with associated methanol molecule, which reduces the νV=O to 848 cm–1 appears to be polymeric with ····V=O····V=O···· linkages and a distorted octahedral coordination geometry in the solid state. This complex displayed all three bands predicted for VO2+ complexes in the visible region in both solvents. This is due to dissociation of the complex in solution to form five-coordinate species. A facile interaction of DMSO with vanadium is indicated by the reduction in the molar absorptivity of band II in this solvent. The above spectral characteristics indicate that VOL16 is six-coordinate in solid and in 96
DMSO. It may be concluded that this complex also exhibit six-coordinate geometry in CHCl3 due to the observation of band I transition and it appeared that the ····V=O····V=O···· linkages was not broken in this solvent. Complex VOL17exhibits only band III at 22 422 cm−1 in CHCl3 and two bands at 15 649-22 624 cm−1 in DMSO. It is difficult, on the strength of this result, to deduce the absolute stereochemistry of this complex but it appeared to exhibit sixcoordinate geometry in DMSO and five-coordinate in CHCl3. The bands are assigned as follows: b2→b1*: 14 728 (CHCl3), 13 850–15 649 (DMSO); b2→eπ*: 16 694–19 685 (CHCl3), 16 891–18 762 (DMSO) and b2→a1*: 22 422–24 876 (CHCl3), 22 624–25 445 (DMSO). The bands in the region 31 348–39 370 cm−1 are attributed to charge-transfer or intra-ligand transitions.
4.4.2.5 Ethoxysalicylaldiimino complexes The
solutions
spectra
of
this
series
are
similar
to
those
of
methoxysalicylaldiimino complexes discussed above. Two d-d transitions were observed in the solution spectra of this series in the visible region except VOL19 which is characterized by a single band in DMSO. Transition corresponding to band I was absent in the solution spectra in both solvents. A shift in band II with a slight lowering in the molar absorptivities of complexes VOL18 and VOL19 is indicative of six-coordinate geometry in DMSO but five-coordinate geometry in CHCl3. A slight shift was observed in the spectrum of VOL20 moving from CHCl3 to DMSO. This indicates different stereochemistry in both solvents. The bands are assigned as follows: b2→eπ*: 16 051–16 694 (CHCl3), 16 155 –17 065 (DMSO) and b2→a1*: 22 831–25 510 (CHCl3), 23 641 (DMSO). Some charge-transfer or intra-ligand bands were observed at 26 110–40 984 cm-1.
97
4.5 Magnetic moment of the metal(II) complexes 4.5.1 Unsymmetrical Schiff base complexes of cobalt(II) The observed values of magnetic moment for cobalt(II) complexes are generally diagnostic of the coordination geometry about the metal ion. The room temperature magnetic moments of low-spin square-planar cobalt(II) complexes are between
1.9−2.9 BM, arising from one unpaired electron plus an apparently large
orbital contribution. Both tetrahedral and high-spin octahedral cobalt(II) complexes possess three unpaired electrons but may be distinguished by the magnitude of the deviation of effective magnetic moment from the spin-only value. The octahedral and tetrahedral cobalt(II) complexes are reported to have magnetic moments between 4.9−5.2 and 4.2−4.8 BM. respectively [39]. The effective magnetic moments of all the cobalt(II) complexes reported here lie in the range of 2.23–2.61 BM. (Table 4.5.1), corresponding to one unpaired electron for square-planar stereochemistry around d7 cobalt(II) ion [180].
4.5.2 Unsymmetrical Schiff base complexes of nickel(II) Nickel(II) has the electronic configuration 3d8 and should exhibit a magnetic moment higher than expected for two unpaired electrons in octahedral (2.8–3.2 BM) and tetrahedral (3.4–4.2 B.M.) complexes. Deviations from spin-only moment of 2.83 BM. due to two unpaired electrons are attributed to orbital contribution. Nickel(II) square-planar complexes are generally diamagnetic [177]. The observed zero magnetic moments confirm the square-planar environment for the nickel(II) complexes in conformity with the fact that all known square-planar complexes of nickel(II) are diamagnetic (Table 4.5.1).
98
4.5.3 Unsymmetrical Schiff base complexes of copper(II) The room temperature magnetic moments of copper(II) complexes are expected to be higher than the spin-only value of 1.73 BM. as a result of orbital contribution and spin-orbit coupling, which mixes in the higher T terms into the ground term. Consequently a magnetic moment of 1.7–2.2 BM. is usually observed for mononuclear copper(II) complexes, regardless of stereochemistry. The magnetic moments of 1.56–2.20 BM. observed for the copper(II) complexes is assigned to four-coordinate, square-planar geometry [60] (Table 4.5.1). The low magnetic moment of CuL2 can be attributed to the possible dimeric structure in which two CuCu atoms interact [183].
4.5.4 Symmetrical and unsymmetrical Schiff base complexes of oxovanadium(IV) The oxovanadium(IV) ion belongs to the S=½ system. The magnetically dilute oxovanadium(IV) complexes usually exhibit magnetic moments corresponding to their spin-only value of 1.73 B.M. The observed magnetic moments of the complexes under study are found in the range 1.60-1.84 BM. and they are presented in Table 4.5.2.
4.6 EPR spectra of oxovanadium(IV) complexes EPR spectroscopy is a useful technique as it provides information on the stereochemistry, ligand type and degree of covalency of oxovanadium(IV) complexes. The unpaired electron responsible for EPR spectrum is confined largely to the oxovanadium(IV) centre. When this unpaired electron interacts with the nuclear spin of
51
V (I = 7/2), the result is (2I+1) or 8 lines separated by coupling
constants with different intensities. The EPR spectra of the oxovanadium(IV)
99
complexes were measured using a Bruker EMX Micro Premium X Spectrometer at X-band of 9.4 GHz on the powder and on the fluid and frozen solutions in toluene/dichloromethane (90:10 v/v). The Spin Hamiltonian parameters were obtained from the simulation of the spectra using a commercial Bruker X Sophe program and all the data are reported in Table 4.6.1-4.6.2 The EPR experimental and simulated spectra are depicted in Figure 4.6.1-4.6.2. Symmetry also has an effect on EPR spectra. The point symmetry at the metal determines whether or not any of the principal values of g or of A are required to be equal to each other and it also determines whether or not any of the principal axes of g and A are required to be coincident. If the spectra are obtained from frozen solutions or as a powder, where the anisotropy is not averaged away by motion of the molecule, a complex pattern can emerge. For example, the electronic configuration around a metal ion may have a unique axis of symmetry. In the commonly employed first derivative display, two superimposed spectra with different hyperfine splittings will be obtained from frozen solutions (‘axial symmetry’). If all three axes in the molecular frame of reference are electronically distinct, three different splittings may be obtained (‘rhombic symmetry’). Due to the strong vanadium–oxygen interaction in the oxovanadium unit, axial or nearly axial EPR spectra are usually observed for oxovanadium(IV) complexes. The parallel transitions, (representing the orientation of the V=O bond parallel to the applied magnetic field), exhibit hyperfine coupling constants that are larger than those for the perpendicular orientation.
100
4.6.1 Unsymmetrical Schiff base complexes of oxovanadium(IV) The EPR spectra of the compounds under consideration were obtained as solid and in toluene-dichloromethane (9:1, v/v) solutions, at room temperature and at 120 K. The powder spectra of the complexes at room temperature and 120 K show a broad single line around g = 2. The g values are all very close to the spin-only value (free electron value) of 2.0023, suggesting minima spin-orbital coupling. The fluid solution spectra of the complexes at room temperature exhibit eight equally spaced lines due to the hyperfine interaction of the unpaired electron with the vanadium nucleus (51V, I = 7/2). The frozen solution spectra of the complexes at 120 K display axial vanadium(IV) spectra (gz = g║ < g┴ = gx = gy and Az = A║ > A┴ = Ax = Ay). The g values, go = 1.971, g║ = 1.978 and g┴ = 1.950, are essentially the same for all the complexes examined. The vanadium nuclear hyperfine splitting, Ao = 101-99, A║ = 65-64, A┴ = 179-177, vary slightly with substituents on the salicylaldehyde. The EPR data for these complexes are in agreements with previously published data for similar complexes with tetradentate bis(Schiff base) ligands [184]. The Ao values are satisfactorily matched with Aav = 99.7-100.7 in all cases, indicating that the configuration of the complexes in solution at room temperature is the same as in frozen state at 120 K. The orders g║ ˂ g┴ and A║ ˃ A┴ are consistent with the oxovanadium(IV) square pyramidal complexes with a C4v symmetry and with the unpaired electron reciding in the dxy orbital [159].
4.6.2 Symmetrical Schiff base complexes of oxovanadium(IV) Five out of the eleven symmetrical oxovanadium(IV) complexes analysed exhibit axial EPR symmetry (gz = g║ < g┴ = gx = gy and Az = A║ > A┴ = Ax = Ay) while four of the complexes exhibit rhombic EPR symmetry (gx ≠ gy≠ gz and Ax ≠ Ay≠ Az).
101
Complex VOL13 was not measured as fluid solution because it did not dissolve in the mixed solvent used. Two of the complexes, VOL17 and VOL19, did not show any hyperfine interaction and they were not simulated. The powder spectra of the complexes at room temperature and 120 K show a broad single line around g = 2. The fluid solution spectra of the complexes at room temperature exhibit eight equally spaced lines due to the hyperfine interaction of the unpaired electron with the vanadium nucleus (51V, I = 7/2). The frozen solution spectra of the complexes VOL5VOL7, VOL9 and VOL12 at 120 K display axial vanadium(IV) spectra with hyperfine structure where g┴ ~ 1.98 with A┴ ~ 60 G and g║ ~ 1.95 with A║ ~ 179 G (gz = g║ < g┴ = gx = gy and Az = A║ > A┴ = Ax = Ay). The Spin Hamiltonian parameters are reported in Table 4.5.1. The EPR experimental and simulated spectra are reported in Figure 4.5. The g║< g┴ and A║ > A┴ relations are consistent with square pyramidal complexes with C4V symmetry with the unpaired electron in the dxy orbital [159]. The frozen solution spectra of the complexes VOL10, VOL14 and VOL19 at 120 K display rhombic vanadium(IV) spectra with three g (gz < gx < gy) and three A values (Az > Ax > Ay) characteristic for C2V symmetry [185]. Usually, this is due to a distortion of the square-pyramidal geometry towards a trigonal-bipyramidal, with the two longer bonds in the axial direction and the three shorter ones in the equatorial plane. Simulation gave the following parameters: gx = 1.976, gy = 1.978-1.980, gz = 1.948-1.950 and Ax = 63.0-64.0, Ay = 58.0-60.0, Az = 178.0-179.0. The Hamiltonian parameters obtained for the oxovanadium(IV) complexes with rhombic symmetry are presented in Table 4.5.2.
102
4.7 Cyclic voltammetry The cyclic voltammetric data for all the complexes are presented in Fig. 4.7 and Table 4.7. At a scan rate of 100 mV/s on a 10–3 M solution of each complex in DMSO containing 0.1 M tetrabutylammonium perchlorate as supporting electrolyte, all the complexes display one well defined oxidation-reduction wave at positive potentials. In an ideal reversible process the peak-to-peak separation, ∆Ep ≈ 59 mV and 30 mV for a one-electron and two-electron processes respectively and ia/ic ≈ 1 for a reversible process. In our complexes ∆Ep = 78±1.8 mV and ia/ic = 0.94±0.04 (approximately 1). Because of the deviation in ∆Ep, which is greater than 59 mV in all the complexes but the ia/ic being approximately 1, we conclude that these complexes are electrochemically pseudo-reversible. The complexes appear to remain stable on a prolonged storage as no significant difference was observed in the voltammogram of VOL12 when it was rerun 24 h after the first reading on the same solution, stored in an air-tight container. The positive E½ values of 401-480.5 mV may be assigned to metal-centred oxidation of VIV to VV in which there appears not to be any change in the structure of parent complex when oxidized [186] and falls within the range reported in the literature for similar complexes [187]. The electrode process can therefore be represented as: [VOIVL]0
[VOVL]+
4.8 Thermal analysis of oxovanadium(IV) complexes Differential scanning calorimetry (DSC) gives information about thermal stability, melting, crystallization, decomposition, desolvation, sublimation, and glass transition. Any reaction or transformation involving absorption or release of heat can also be detected with this technique. The thermal characteristic data of the 103
complexes were determined from DSC thermograms (melting points, taken as the temperature corresponding to the minimum of the endothermic peak) and the results are summarized in Table 4.8 and Figure 4.8.1.1-4.8.2.5.3. The analysis of the DSC thermograms indicates that all the oxovanadium(IV) complexes under study are thermally stable at constant ambient pressure between 25 and 300 oC. The DSC analyses were carried out mainly to determine the melting and the decomposition temperatures of the oxovanadium(IV) complexes because their melting points were outside the range covered by the melting point instrument.
4.8.1 Unsymmetrical Schiff base complexes of oxovanadium(IV) Four peaks were observed in the DSC thermogram of VOL1, corresponding to three different endothermic peaks followed by an exothermic peak representing the decomposition of the complex (Fig. 4.8.1.1). The complex showed some morphology transformations [188] between 350 and 365 oC, followed by the sharp endothermic peak at 385
o
C corresponding to the melting point of the complex. A close
examination of the DSC peaks show that there is a hump near the onset temperature of the exothermic peak. This may be due to the competition between the melting and decomposition processes while the latter process predominates. The exothermic peak at 455 oC corresponds to the decomposition of the complex. The DSC curve of VOL3 (Fig. 4.8.1.3) shows a sharp endothermic peak with a minimum at 310 oC corresponding to the melting process. Immediately after melting, decomposition occurs and a broad exothermic peak was observed at 365 oC. VOL4 gave a moderate exothermic peak at 455 oC, after an endothermic peak at 335 oC (Fig. 4.8.1.4), corresponding to the melting point of the complex. The hump observed near the onset temperature of the exothermic peaks of both VOL3 and VOL4 is
104
indicative of competition between the melting and decomposition processes while the latter process predominates. The DSC curve (Fig. 4.8.1.2) of VOL2 showed no melting but a sharp exothermic decomposition at 380
o
C. Thus this complex
decomposed without melting.
4.8.2 Symmetrical Schiff base complexes of oxovanadium(IV) 4.8.2.1 Benzophenoneimino complexes The DSC thermogram of VOL5 (Fig. 4.8.2.1.1) shows a sharp endothermic peak with a minimum at 401 oC corresponding to the melting point of the complex. There was no exothermic peak observed which indicates that the complex did not decompose up to 500 oC. Two endothermic peaks were observed in the thermogram of VOL7. The DSC curve (Fig. 4.8.2.1.3) presented an endothermic peak (broad) which we provisionally assign to the loss of the MeOH, followed by a melting process at 345 oC represented by a sharp endothermic peak.
4.8.2.2 Naphthaldiimino complexes The DSC thermograms of the members of this series all show single sharp endothermic peaks at 390, 330 and 420 oC (Fig. 4.8.2.2.1-4.8.2.2.3). These peaks correspond to the melting points of the complexes VOL8, VOL9 and VOL10 respectively. The melting points of the members of this series do not correspond to the order of their molecular masses.
4.8.2.3 Chlorosalicylaldiimino complexes The DSC thermograms of the complexes VOL11 and VOL13 show one sharp endothermic peak and one exothermic peak. It can be observed that the
105
melting and the onset of the decomposition are partially superposed (Fig 4.8.2.3.1 and 4.8.2.3.3). The endothermic peaks which correspond to their melting points has its minimum at 420 and 415 oC while the exothermic peaks occur at 430 and 425 oC respectively. The DSC curve of complex VOL12 (Fig. 4.8.2.3.2) shows three peaks, two of the peaks correspond to an exothermic processes and one corresponds to an endothermic process. The sharp endothermic peak at 315 oC corresponds to melting which is followed by broad exothermic peaks at 360 and 395
o
C; the peaks
immediately after the melting is due to stepwise decomposition of the complex. The DSC thermogram of VOL14 (Fig. 4.8.2.3.4) shows a sharp endothermic peak at 460 o
C corresponding to the melting point of the compound.
4.8.2.4 Methoxysalicylaldiimino complexes The DSC thermogram of VOL17 shows three peaks, the weak endothermic peak at about 120 oC is due to morphological transformation, while the sharp endothermic peak at 345 oC corresponds to melting. This is followed by a strong exothermic peak at 415 oC. In the DSC curve of VOL16 (Fig. 4.8.2.4.2), a sharp endothermic peak was observed at about 348
o
C. This was followed by
decomposition process which corresponds to a broad exothermic peak with the maximum at 390 oC. In the DSC curve of VOL15, a sharp endothermic peak, with the minimum at 325 oC corresponding to the melting process was observed. A broad exothermic peak at 440 oC is observed in the thermogram of the complex which corresponds to the decomposition procress.
106
4.8.2.5 Ethoxysalicylaldiimino complexes An endothermic peak at 240 oC was observed in the thermogram of complex VOL19 which corresponds to its melting point. The exothermic peak at 360 oC represents the decomposition of the complex (Fig. 4.8.2.5.2). Three peaks were observed in the DSC curve of VOL18, two endothermic peaks and one exothermic peak. The first endothermic peak might be an evaporation process because the complex did not melt when heated to 250 oC and this peak is observed at 245 oC. The second endothermic peak at 290 oC corresponds to the melting point of the complex (Fig. 4.8.2.5.1). The exothermic peak is observed at about 360 oC which corresponds to the decomposition of the complex. The DSC thermogram of VOL20 (Fig. 4.8.2.5.3) shows two sharp peaks and two broad and weak peaks. The two weak and broad endothermic peaks were between 75 - 140 oC and they are due to morphological transformations [188]. A sharp endothermic peak with minimum at 310 oC corresponds to the melting point of the complex. The complex decomposed at about 375 oC. In general, the DSC thermograms of the complexes reveal a sharp endothermic process corresponding to sharp melting points followed by an exothermic decomposition processes. Few of the complexes show some weak endothermic peaks before the melting processes and these peaks are due to morphological transformation in the complexes.
107
CHAPTER 5 INSULIN-MIMETIC STUDIES ON THE OXOVANADIUM(IV) COMPLEXES
5.1 In vitro analysis The use of in vitro studies to evaluate the glucose uptake in cell lines following stimulation with insulin and other active compounds is a direct and sensitive method of determining the antidiatogenic effect of these compounds [189, 190]. It allows for rapid screening of compounds in terms of their efficacy and toxicity in an ethically acceptable manner. Although the in vitro environment does not accurately mimic the in vivo situation, it offers unique opportunities and insights into the biochemical pathways which control the utilization of glucose by different cell types following exposure to various agents with potential diabetogenic effects. At the cellular level glucose uptake is mediated through glucose transporters. Glucose transporter subtype 4 (GLUT4) is the insulin-responsive transporter of glucose in various cell types in the body [189, 190]. Muscle and fat derived cell lines including C2C12 and L6 myoblast have been shown to be sensitive to insulin stimulation in culture resulting in an increase of GLUT4 and glucose uptake from the culture medium [189, 190]. Metformin and insulin were included in this analysis for comparative purposes. Metformin is an oral anti-diabetic drug from the biguanide family. It is the first-line drug for the treatment of type 2 diabetes, particularly in overweight and obese people and those with normal kidney function. Metformin improves hyperglycemia primarily through its suppression of hepatic glucose production (hepatic gluconeogenesis). In addition to suppressing hepatic glucose production, metformin increases insulin sensitivity, enhances peripheral glucose uptake, increases fatty acid oxidation [191], 108
and decreases absorption of glucose from the gastrointestinal tract. Increased peripheral utilization of glucose may be due to improved insulin binding to insulin receptors [192]. AMP-activated protein kinase (AMPK) probably also plays a role, as metformin administration increases AMPK activity in skeletal muscle [193]. AMPK is known to cause GLUT4 translocation, resulting in insulin-independent glucose uptake. C2C12 cells were pre-exposed to the oxovanadium(IV) compounds, insulin and metformin respectively in glucose and serum-free media for 3 h before the glucose uptake experiments. Basal glucose uptake i.e. solvent vehicle only (DMSO) is represented as 100% and the subsequent increase or decrease induced by the compounds is reflected as ± 100%. Cytotoxic test carried out on all the complexes using 3-(4, 5-dimethylthiazo)-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay showed that the complexes were not toxic to the cells at both low (0.05 μL) and high (0.25 μL) concentrations.
5.1.1 Unsymmetrical Schiff base complexes of oxovanadium(IV) The insulin-like capacity of vanadium compounds is usually related to their ability to lower the blood glucose level by activating the glucose transport into the cell of the peripheral tissues. In this study, the in vitro glucose uptake by C2C12 muscle cells following exposure to four unsymmetrical tetradentate Schiff base complexes of oxovanadium(IV) has been investigated. Insulin-mimetic test on C2C12 muscle cells shows that all the complexes significantly stimulated cell glucose uptake when compared to the basal glucose uptake of the solvent vehicle (DMSO) with negligible cytotoxicity at the concentration of 0.05 μg/mL (Figure 5.1.1 and Table 5), but not at the same levels as insulin and metformin.
109
It seems that substitution on the salicylaldehyde group lowers the insulin enhancing activity as VOL4 gave the largest effect. It was also observed that the electronic state of the substituents has some effects on the effectiveness. The order of activity, VOL4 > VOL1 ~ VOL3 > VOL2 correspond to H, Cl (-I, +M), OEt (+I, +M), NO2 (-I, -M) substituents on the salicylaldehyde ring. The lowest percentage glucose utilization for complex VOL2 may therefore be attributed to the greater negative electron withdrawing effect (-I and –M) of the nitro group on the salicylaldehyde when compared to the other substituents.
5.1.2 Symmetrical Schiff base complexes of oxovanadium(IV) 5.1.2.1 Benzophenoneimino complexes All tested complexes in the series increased glucose utilization in C2C12 cells over basal values except VOL6 whose percentage glucose uptake was lower than the basal glucose uptake (DMSO). Two of the complexes VOL5 and VOL7 significantly increased glucose uptake (p=0.001) over basal values. VOL7, which shows the highest activity when compared to the other members of this series, could be attributed to the presence of an adduct (MeOH) in the molecule. The percentage glucose uptake of VOL7 was comparable close to that of metformin as shown in Fig. 5.1.1 and Table 5.
5.1.2.2 Naphthaldiimino complexes All the complexes increased glucose uptake in C2C12 cells over basal glucose uptake. Complex VOL8 significantly increased glucose uptake (p= 0.001) when compared to the basal glucose uptake (DMSO). The ethylenediamine-bridged complexes in the whole series studied showed some increase in activity when
110
compared to the other diamines used. The results are presented in Table 5 and Fig. 5.1.1.
5.1.2.3 Chlorosalicylaldiimino complexes All tested complexes in the series significantly increased glucose uptake in C2C12 cells if compared to the basal glucose uptake (DMSO). It seems from the above observation that the +M and –I effect of the chlorine atom plays an important role in the increased activities of the complexes in this series as shown in Fig. 5.1.2 and Table 5.
5.1.2.4 Methoxysalicylaldiimino complexes All the complexes tested in the series also significantly increased glucose uptake if compared to the basal glucose uptake (solvent vehicle). The percentage glucose uptake of VOL18 was higher than that of metformin but less than that of insulin (Fig. 4.9.2 and Table 4.9). The complex VOL16 showed a remarkable glucose uptake activity in that its percentage glucose uptake was higher than those of insulin and metformin. The activities of this series may be attributed to the presence of the methoxy substituent on the salicylaldehyde because the same set of diamines that was used in the other series was also used in this series.
5.1.2.5 Ethoxysalicylaldiimino complexes Two of the complexes increased glucose consumption in C2C12 cells over basal values but VOL20 recorded a lower percentage glucose uptake than the basal glucose uptake (DMSO). The presence of the ethoxyl substituent on the salicylaldehyde did not seem to affect the activities of this complex as much as the
111
methoxyl group did in the series above but complex VOL18 significantly increased glucose (p= 0.001) in C2C12 cells if compared to the basal glucose uptake as shown in Fig. 5.1.2 and Table 5.
5.2 In vivo analysis Vandate(V) and oxovanadium(IV) compounds have been well documented to mimic many of the actions of insulin [77]. Vanadium compounds have been shown to increase glucose transport and oxidation, to stimulate glycogen synthesis in the liver and to inhibit gluconeogenesis, i.e., vanadium is able to mimic most of the biological effects of insulin in various cell types[92]. Great efforts have therefore been made to prepare vanadium(IV) and vanadium(V) complexes of high biological activity and low toxicity which are readily absorbed. Many oxovanadium(IV) complexes with various coordination modes like VO(O4), VO(S2N2), VO(S4), VO(N4) and VO(N2O2),[93, 132] have been prepared, and the relationship between their structures and insulinmimetic activities has also been examined by evaluating both in vitro and in vivo experimental results. As mentioned in Chapter 1, VO(maltolate)2, VO(picolinate)2, and VO(6-Me-picolinate)2 proved to be potent in decreasing the blood glucose level with high efficiency. A detailed literature review show that till date, only [VO(sal2-en)] [126] and [VO(sal2-1,3-pn)] [127] among complexes of the type reported in this work have been tested for insulin mimetic.
[VO(sal2-en)] was reported to be orally effective for
glucose lowering in alloxan-induced diabetic rats, with a tendency for rats to be hypoglycemic; withdrawal of treatment results in the reversal to hyperglycemia. On the other hand [VO(sal2-1,3-pn)] normalized glucose and lipid values without an increase in insulin levels, and improved glucose tolerance.
112
In this study, rats were injected with STZ to reduce or deplete the number of B cells to induce an insulin deficient non-ketoacidotic hyperglycaemia at levels typical of a stable Type 1, or late stage Type 2 diabetes. This is to test the effect of oxovanadium(IV) complexes in reducing fasting glucose levels in the absence (T1D) or presence of a compromised insulin production (late stage T2D). If the complexes do show a lowering effect of blood glucose it might be possible that the complexes have an insulin-like effect or extra-pancreatic actions. The extra-pancreatic actions could be stimulation of peripheral glucose utilization or enhancing glycolytic and glycogenic processes with a decrease glycogenlysis and gluconeogenesis. Nine of the twenty oxovanadium(IV) complexes were tested in vivo for their insulin mimetic activities. An acute oral administration of the four unsymmetrical Schiff base complexes of oxovanadium(IV) elicited a progressive reduction in plasma glucose over 6 h in STZ rats. Two of the unsymmetrical Schiff base complexes of oxovanadium(IV) induced a significant reduction in plasma glucose over a 6 h period.
5.2.1 Unsymmetrical Schiff base complexes of oxovanadium(IV) An acute oral administration of the unsymmetrical complexes at a dose of 0.2 mmol/kg to STZ rats elicited a progressive reduction in plasma glucose over 6 h periods.
Acute oral administration of VOL1 and VOL4 to STZ rats induced a
significant reduction in plasma glucose over a 6 h period. The complex VOL1, significantly reduced plasma glucose by 20.48 % after 6 h (p = 0.04) while complex VOL4 also significantly reduced plasma glucose by 28.87 % at 5 h (p = 0.03) and by 31.91 % at 6 h (p = 0.007). The percentage reduction in plasma glucose after treatment of the STD rats with the complexes is shown in Fig. 5.2.1
113
The above results confirm the structural activities proposed under the in vitro analysis, that substitution on the salicylaldehyde ring lowers the insulin enhancing activity as VOL4 also gave the largest effect in the in vivo analysis. Almost the order of activity, VOL4 > VOL1 > VOL2 > VOL3 corresponding to H, Cl (-I, +M), OEt (+I, +M), NO2 (-I, -M) substituents on the salicylaldehyde ring proposed under the in vitro discussion was observed in the in vivo experiments. These results demonstrate that oral administration of the complexes is effective in lowering diabetic hyperglycemia.
5.2.2 Symmetrical Schiff base complexes of oxovanadium(IV) An acute oral administration of the complexes VOL5, VOL6, VOL7, VOL12 and VOL13 elicited a progressive reduction in plasma glucose over 6 h in STZ rats. Acute oral administration of VOL7 and VOL13 to STZ rats induced a significant reduction in plasma glucose during the 6 h period. Complex VOL7 significantly reduced plasma glucose by 26.23 % (p=0.01) after 5 h and by 24.56 % (p=0.05) after 6 h. Complex VOL13 was found to significantly reduced plasma glucose by 10.58 % (p=0.03) after 1 h. The electronic effect of the chlorine substituent (-I, +M) on the salicylaldehyde, like in the in vitro studies, might be responsible for the high activity of VOL13. The percentage reduction in plasma glucose after treatment of the STD rats with the complexes is shown in Fig. 5.2.2. The optimal time-course procedure used in this study to investigate the short-term insulin-mimetic effect of these complexes showed a significant hypoglycaemic effect from 0 to 6 h when compared to the control. From the results obtained from the in vitro and in vivo analyses, it may be inferred that the complexes under observation will also enhance glucose utilization or lowering of glucose serum level if they are administered over a long-term period.
114
It is very difficult to compare the efficacy of insulin-mimetic activities of vanadium complexes in published studies unless the studies are carried out for the specific purpose of comparison. These are due to variables such as method of administration, dose, diet, liquid consumption, gravity of the disease and the genetics, metabolism and environment of the animal model. Comparison and evaluation can therefore only be done if a reference compound has been included in the study for the purpose of comparison. BMOV has been experimentally used as a representative standard in several biological studies due to its approval for clinical experimient [194]. But BMOV was not included in this study because the pharmacological beneficial dose is associated with some toxicity and hence attempts are underway to evolve vanadium complexes with fewer side effects while retaining their enhanced therapeutic activities.
115
CHAPTER 6 CONCLUSIONS AND FUTURE PROSPECTS 6.1 Conclusions The involvement of azomethine N and phenolic O of the tertadentate Schiff bases to the metal ions was confirmed by comparing the IR data of the ligands with those of the metal(II) complexes. Further conclusive evidence of the coordination of these Schiff-bases with the metal ions was shown by the appearance of new bands due to ν(M―N) and ν(M―O) in the metal complexes. The unsymmetrical nature of the unsymmetrical Schiff bases and their metal(II) complexes was confirmed by their IR spectra where two bands were observed for each of ν(C=N), ν(C―O), ν(V―N) and ν(V―O), taking their origin from the different aldehydes contained in each ligand. Most of the oxovanadium(IV) complexes exhibit a strong band in the range 959−989 cm−1, which have been assigned to ν(V=O) in a monomeric square pyramidal environment. The oxovanadium(IV) complexes with trimethylene bridge, in which the ν(V=O) appeared at 848−860 cm–1, have been assigned polymeric structure with ····V=O····V=O···· interactions, which afforded distorted octahedral coordination geometry. The electronic spectral and magnetic susceptibility measurements were used for assigning the stereochemistry of each metal(II) complexes. For four-coordinate metal(II) complexes, either a square planar or a tetrahedral configuration is possible. On the basis of electronic spectra, together with the magnetic moments, it was possible for us to assign the preffered configuration for the metal(II) complexes reported herein. The electronic spectra indicate a low spin square-planar geometry for all the cobalt(II) complexes. This was also corroborated by the effective magnetic moment of 116
the complexes, which lie in the range of 2.23–2.61 B.M., corresponding to one unpaired electron for low spin square-planar cobalt(II) complexes. The appearance of a single d−d transition in DMSO is attributed to solvent effect, indicating the possibility of formation of five- or six-coordinate species. A square-planar geometry was assigned to all the red nickel(II) complexes. The observed zero magnetic moment confirms the square-planar environment in conformity with the fact that all known square-planar complexes of nickel(II) are diamagnetic. The stereochemistry of these complexes was unchanged in coordinating solvent (DMSO), except for NiL2 containing nitro group on the aromatic ring, where some changes were observed even though the observed bands in the complex were still in conformation with a squre-planar geometry. For the copper(II) complexes, a distorted square-planar N2O2 coordination chromophore was assigned. The distorted square-planar geometry of these complexes was also supported by their effective magnetic moments in the range 1.56–2.20 B.M. The low magnetic moment value of CuL2 can be attributed to possible dimerization in the complex. A multiple spectroscopic techniques were used to established the stereochemistry of the oxovanadium(IV) complexes. The electronic spectra of the oxovanadium(IV) complexes suggest a diversity of geometries. The electronic spectra indicate a square-pyramidal geometry for the five-coordinate species and distorted octahedral geometry for the six-coordinate species. The room temperature magnetic moments of 1.6–1.8 B.M. are normal for V(IV) d1 configuration. In chloroform, the complexes are most probably five-coordinate with square-pyramidal geometry. The observed shift in band II of most these complexes with a lowering of
117
intensity in the molar absorptivity from CHCl3 to DMSO are indicative of sixcoordinate geometry in DMSO. The frozen solution EPR spectra of the complexes VOL10, VOL14, and VOL19 at 120 K display rhombic vanadium(IV) spectra with three g (gz < gx < gy) and three A values (Az > Ax > Ay) characteristic for C2V symmetry. Usually, this is due to a distortion of the square-pyramidal geometry towards a trigonal-bipyramidal. The solution EPR spectra of the other oxovanadium(IV) study are consistent with square pyramidal complexes with C4v symmetry. The cyclic voltammetry revealed only one quasi-reversible wave for each oxovanadium(IV) complex and they all showed redox couples with peak-to-peak separation values ranging from 74 to 83 mV, indicating a single step one-electron transfer process. In vitro glucose uptake was carried out on all the oxovanadium(IV) complexes using C2C12 cell line. All the complexes tested increased glucose utilization in C2C12 cells over basal values except two of the complexes whose percentage glucose uptake was lower than the basal glucose uptake (DMSO). Eighteen of the oxovanadium(IV) complexes significantly increased glucose uptake when compared to the basal glucose uptake of the solvent vehicle (DMSO). Cytotoxic test carried out on all the complexes using MTT assay showed that the complexes were not toxic to the cells at both low and high concentrations. Two of the complexes showed activities comparable or greater than that of insulin. Among the symmetrical oxovanadium(IV) complexes subjected to in vitro studies, the methoxysalicylaldiimines and chlorosalicylaldiimines complexes showed remarkable glucose uptake activity in that their percentage glucose uptake were higher than basal glucose uptake. All the members of the two series significantly
118
increased glucose uptake in C2C12 cells on comparison to the basal glucose uptake. We have proposed that the +M and –I effects of the chlorine and the +M and +I effects of the methoxy group play important roles in the enhanced activities of this series. It was also observed that all the ethylenediamine-bridged complexes showed enhanced activities. Four unsymmetrical and five symmetrical Schiff base complexes of oxovanadium(IV) have been tested in vivo for their insulin mimetic activities. An acute oral administration of the four unsymmetrical Schiff base complexes of oxovanadium(IV) elicited a progressive reduction in plasma glucose over 6 h in STZ rats. Two of the unsymmetrical Schiff base complexes of oxovanadium(IV) induced a significant reduction in plasma glucose over a 6 h period. Oral administration of the five symmetrical complexes also elicited a progressive reduction in plasma glucose over 6h. Two of these complexes induced a significant reduction in plasma glucose during the 6 h period. The in vitro and in vivo studies carried out on the unsymmetrical complexes show structure-activity relationships brought about by the substituents on the salicylaldehyde group. The results show that substitution on the salicylaldehyde group lowers the insulin enhancing activity of the complexes as the complex without substitution gave the largest effect.
6.2 Suggestion for future work Novel
symmetrical
and
unsymmetrical
Schiff
base
complexes
of
oxovanadium(IV) suitable for insulin-enhancing biological studies were successfully synthesized. The results from this work show that most of the complexes are promising insulin-enhancing agents. To get a better understanding of the insulinmimetic properties of these compounds, further studies would need to be carried out
119
to determine their mechanism of action. In vitro and in vivo glucose uptake studies should be carried out using the radioactive 2-deoxyglucose (DOG) to monitor their mode of actions. Furthermore, all the complexes need to be screened for the antimicrobial properties using different strain of micro organisms to explore other areas of possible application. The synthetic methodology for preparation of the unsymmetrical Schiff bases (Chapter three) will find application in the synthesis of more unsymmetrical Schiff bases with more substituents on the aromatic 2-hydroxyaldehydes. There is therefore room for the synthesis of more unsymmetrical Schiff bases and their metal complexes and further exploration of their biological activities. The need to obtain good crystals for structural studies, lacking in this work, cannot be over-emphasized.
120
References [1] J. Hine, C.Y. Yeh, Equilibrium in formation and conformational isomerization of imines derived from isobutyraldehyde and saturated aliphatic primary amines, J. Am. Chem. Soc. 89 (1967) 2669. 2] R.J. Fessenden, J.S. Fessenden, Organic Chemistry, Brooks/Cole Publishing Company, USA, 1998. [3] Z. Cimerman, S. Miljanic, N. Galic, Schiff bases derived from aminopyridines as spectrofluorimetric analytical reagents, Croatica Chemica Acta 73 (1) (2000) 81. [4] A. Elmali, M. Kabak, Y. Elerman, Conformational study and structure of bisN,N′-p-bromo-salicylideneamine-1,2-diaminobenzene, J. Mol. Struct. 477 (2000) 151. [5] M. Valcarcel, M.D. Laque de Castro, Flow-throgh Biochemical Sensors, Elsevier, Amsterdam., 1994. [6] U. Spichiger-Keller, Chemical Sesors and Biosensors for Medical and Biological Applications, Wiley-VCH, Weinheim, 1998. [7] J.F. Lawrence, R.W. Frei, Chemical Derivatization in Chromatography, Elsevier, Amsterdam, 1976. [8] S. Patai (Ed.), The Chemistry of the Carbon-Nitrogen Double Bond, J. Wiley & Sons, London, 1970. [9] E. Jungreis, S. Thabet, Analytical Applications of Schiff bases, Marcell Dekker, New York, 1969. [10] C.M. Metzler, A. Cahill, D.E. Metzler, Equilibriums and absorption spectra of Schiff bases, J. Am. Chem. Soc. 102 (1980) 6075. [11] C. Spinu, M. Pleniceanu, C. Tigae, Biologically Active Transition Metal Chelates with a 2-Thiophenecarboxaldehyde-Derived Schiff Base: Synthesis, Characterization, and Antibacterial Properties, Turk. J. Chem. 32 (2008) 487. [12] B. Clarke, N. Clarke, D. Cunningham, T. Higgins, P. McArdle, M. Ni Cholchu, M. O’Gara, Transition-metal Schiff-base complexes as ligands in tin chemistry. Part 7. Reactions of organotin(IV) Lewis acids with [M(L)]2 [M-Ni, Cu and Zn; H2L-N,N%bis(3- methoxysalicylidene)benzene-1,3-diamine and its -1,4-diamine analog], J. Organometallic Chem. 559 (1998) 55. [13]
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134
APPENDIX I Table 4.1.1 Physical properties and analytical data for the Schiff bases and oxovanadium(IV) complexes Colour
M.P./ oC
Orange -Yellow
194-195
81.6
Green
>250
411.41
86.2
134-135
C24H15N3O5V
476.34
75.4
Orange -Yellow Green
H2L3
C26H22N2O3
410.47
71.9
135-136
VOL3
C26H20N2O4V
475.40
79.2
Orange -Yellow Green
H2L4
C24H18N2O2
366.41
78.4
187-188
VOL4
C24H16N2O3V
431.34
92.8
Orange -Yellow Green
H2L5
C28H24N2O2
420.5
90.7
Yellow
164-165
VOL5
C28H22N2O3V
485.4
73.3
Green
>250
H2L6
C29H24N2O2
434.5
86.5
Yellow
154-155
VOL6
C29H22N2O3V
499.5
83.9
Green
>250
H2L7
C29H26N2O2
434.5
82.2
Yellow
123-124
VOL7
C30H28N2O4V
531.5
67.0
Orange
>250
H2L8
C24H20N2O2
368.4
95.6
Yellow
>250
VOL8
C24H18N2O3V
382.4
93.9
Green
>250
H2L9
C25H22N2O2
382.5
89.2
Yellow
222-223
VOL9
C25H20N2O3V
447.4
95.6
Orange
>250
H2L10
C28H20N2O2
416.5
86.4
Orange
233-234
VOL10
C28H18N2O3V
481.4
72.5
Brown
>250
Compound H2L1
Empirical Formula C24H17N2O2Cl
Formula weight 400.86
Yield (%) 91.5
VOL1
C24H15N2O3ClV
465.79
H2L2
C24H17N3O4
VOL2
135
>250
>250
>250
Microanalysis (Calc.) %C %H %N 71.60 4.27 6.98 (71.83) (4.73) (6.60) 62.22 (61.83) 69.69 (70.07) 60.7 (60.52) 76.34 (76.08) 65.69 (65.69) 78.41 (78.67) 66.29 (66.83) 79.98 (79.95) 69.28 (69.06) 80.16 (80.12 69.74 (69.80) 79.92 (80.16) 67.79 (67.02) 78.24 (78.11) 65.72 (65.17) 78.51 (78.52) 67.12 (67.11) 80.75 (80.55) 69.86 (70.40)
3.15 (3.24) 4.23 (4.16) 3.38 (3.17) 5.51 (5.40) 5.51 (5.40) 4.81 (4.95) 3.86 (3.74) 5.75 (5.77) 4.57 (4.49) 6.03 (6.12) 4.84 (4.83) 5.97 (6.03) 5.31 (5.36) 5.47 (5.49) 4.90 (5.13) 5.80 (5.82) 4.51 (4.41) 4.84 (4.69) 3.77 (3.66)
6.11 (6.01) 10.12 (10.21) 8.67 (8.82) 6.79 (6.82) 5.91 (5.89) 7.62 (7.65) 6.38 (6.49) 6.66 (6.68) 5.77 (5.98) 6.45 (6.48) 5.61 (5.64) 6.41 (6.45) 5.46 (5.29) 7.60 (7.62) 5.68 (5.61) 7.32 (7.37) 6.26 (6.39) 6.73 (6.72) 5.82 (6.03)
H2L11
C16H14N2O2Cl
337.2
88.7
Yellow
178-179
56.84 (57.05)
4.17 (4.13)
8.28 (8.29)
VOL11
C16H12N2O3Cl2V
402.1
91.3
Green
>250
H2L12
C17H16N2O2Cl2
351.2
82.4
Yellow
122-123
47.68 (47.85) 58.14 (57.77)
3.00 (2.87) 4.59 (4.53)
6.95 (7.06) 7.98 (7.83)
VOL12
C17H14N2O3Cl2V
416.2
76.1
Green
>250
H2L13
C17H16N2O2 Cl2
351.2
78.6
Yellow
61-62
VOL13
C17H14N2O3Cl2V
416.2
79.0
Orange
>250
H2L14
C20H14N2O2 Cl2
385.2
84.2
219-220
VOL14
C20H12N2O3Cl2V
450.1
79.5
Orange -Yellow Orange
H2L15
C18H20N2O4
328.4
83.4
Yellow
165-166
VOL15
C20H18N2O5V
397.3
86.7
Green
>250
H2L16
C19H22N2O4
342.4
79.6
Yellow
82-83
VOL16
C20H24N2O6V
443.4
81.5
Orange
>250
H2L17
C22H20N2O4
376.4
65.1
Orange
103-104
VOL17
C22H18N2O5V
441.3
74.3
Brown
>250
H2L18
C20H24N2O4
356.4
80.2
Yellow
138-139
VOL18
C20H22N2O5V
421.4
91.6
Green
>250
H2L19
C21H26N2O4
370.5
89.3
Yellow
70-71
VOL19
C21H24N2O5V
435.4
88.5
Green
239-240
H2L20
C24H24N2O4
404.5
79.7
72-73
VOL20
C24H22N2O5V
469.4
75.5
Orange -Yellow Green
49.07 (48.86) 58.14 (58.00) 49.07 (48.99) 62.31 (62.21) 53.25 (53.25) 65.82 (65.84) 54.85 (54.97) 66.58 (66.65) 55.80 (56.03) 70.23 (70.20) 57.92 (58.34) 67.52 (67.40) 56.93 (57.01) 67.73 (68.09) 57.45 (57.93) 71.43 (71.27) 59.98 (61.41)
3.39 (3.31) 4.59 (4.63) 3.39 (3.34) 3.57 (3.65) 2.59 (2.68) 6.14 (6.14) 4.61 (4.61) 6.50 (6.48) 4.79 (4.95) 5.29 (5.36) 4.60 (4.68) 6.98 (6.79) 5.32 (5.26) 7.15 (7.07) 5.49 (5.56) 5.98 (5.98) 4.93 (4.47)
6.73 (6.52) 7.98 (8.00) 6.78 (6.88) 7.30 (7.25) 6.31 (6.21) 8.51 (8.53) 7.12 (7.12) 8.15 (8.18) 6.82 (6.88) 7.45 (7.44) 5.94 (5.92) 7.96 (7.86) 6.79 (6.65) 7.51 (7.56) 6.40 (6.43) 7.01 (6.93) 5.88 (5.64)
136
>250
>250
Table 4.1.2 Physical properties and analytical data for cobalt(II), nickel(II) and copper(II) the compounds
Complex 1
Empirical Formula
Formula Mass
% Yield
Colour
M.P./ oC
CoL
C24H15N2O2ClCo
458.25
93.2
Brown
>250
CoL2
C24H15N3O4Co
468.34
89.5
Brown
>250
CoL3
C26H20N2O3Co
467.39
94.7
Brown
>250
CoL4
C24H16N2O2Co
423.34
91.8
Brown
>250
NiL1
C24H15N2O2ClNi
458.00
95.8
Red
>250
NiL2
C24H15N3O4Ni
468.10
96.1
Red
>250
NiL3
C26H20N2O3Ni
467.15
90.2
Red
>250
NiL4
C24H16N2O2Ni
423.10
88.8
Red
>250
CuL1
C24H15N2O2ClCu
462.86
90.9
>250
CuL2
C24H15N3O4Cu
472.95
86.3
CuL3
C26H20N2O3Cu
472.01
91.7
CuL4
C24H16N2O2Cu
431.34
94.8
Pale Brown Pale Brown Pale Brown Pale Brown
137
>250 >250 >250
Microanalysis (Calc.) %C 63.17 (62.91) 61.97 (61.55) 66.62 (66.82) 68.31 (68.09) 63.21 (62.94) 61.02 (61.58) 66.49 (66.85) 68.11 (68.13) 62.06 (62.28) 60.72 (60.95) 66.03 (66.16) 66.29 (66.83)
%H 3.20 (3.30) 3.10 (3.23) 4.30 (4.28) 3.65 (3.81) 3.22 (3.30) 3.16 (3.23) 4.17 (4.32) 3.70 (3.81) 3.11 (3.27) 3.47 (3.20) 4.50 (4.27) 3.86 (3.74)
%N 5.81 (6.11) 8.63 (8.97) 5.91 (5.99) 6.16 (6.62) 5.87 (6.12) 8.87 (8.98) 5.51 (6.00) 6.31 (6.62) 5.68 (6.05) 9.13 (8.88) 5.48 (5.93) 6.38 (6.49)
Table 4.2 1H NMR data for free ligands (chemical shifts in ppm) Ligand
δ OH
δ Ar-H
H2L1
6.9-8.1(m,13H)
H2L5
15.5 (s, 1H) 10.2 (s, 1H) 13.9 (s, 1H) 15.2 (s, 1H) 12.9 (s, 1H) 15.0 (s, 1H) 12.0 (s, 1H) 15.7 (s, 1H) 15.2 (s, 2H)
H2L6
δ CH3
δ CH2
δ CH
δ N=CH
1.5 (t, 3H)
4.1 (q, 2H)
6.6-7.5 (m, 18H)
―
3.7 (t, 4H)
―
8.9 (s, 1H) 9.0 (s, 1H) 8.4 (s, 1H) 8.8 (s, 1H) 8.2 (s, 1H) 8.7 (s, 1H) 8.6 (s, 1H) 8.9 (s, 1H) ―
15.3 (s, 2H)
6.6-7.5 (m, 18H)
1.3 (d, 3H)
3.7 (d, 2H)
3.5 (m, 1H)
―
H2L7
15.5 (s, 2H)
6.6-7.5 (m, 18H)
―
―
―
H2L8
14.3 (s, 2H)
7.2-8.0 (m, 12H)
―
2.0 (m, 2H) 3.4 (t, 4H) 4.0 (s, 4H)
―
8.2 (s, 2H)
H2L9
14.8 (s, 2H)
7.0-7.9 (m, 12H)
―
―
8.9 (s, 2H)
H2L10
15.1 (s, 2H)
7.2 -8.1 (m,12H)
―
2.2 (m, 2H) 3.8 (t, 4H) ―
―
9.5 (s, 2H)
H2L11
13.1 (s, 2H)
6.9-7.3 (m, 6H)
―
4.0 (s, 4H)
―
8.3 (s, 2H)
H2L12
13.1 (s, 2H)
6.6-7.3 (m, 6H)
1.3 (d, 3H)
3.9 (d, 2H)
3.7 (m, 1H)
8.3 (s, 2H)
H2L13
13.4 (s, 2H)
6.6-7.3 (m, 6H)
―
―
8.3 (s, 2H)
H2L14
13.0 (s, 2H)
6.9-7.4 (m, 10H)
―
2.1 (m, 2H) 3.7 (t, 4H) ―
―
8.6 (s, 2H)
H2L15
12.7 (s, 2H)
6.7-7.3 (m, 6H)
3.8 (s, 3H)
4.0 (s, 4H)
―
8.3 (s, 2H)
H2L16
12.9 (s, 2H)
6.7-7.3 (m, 10H)
3.8 (s, 3H)
―
8.3 (s, 2H)
H2L17
12.7 (s, 2H)
6.9-7.7 (m, 10H)
3.8 (s, 3H)
3.7 (t, 4H) 2.2 (m, 2H) ―
―
8.6 (s, 2H)
H2L18
13.6 (s, 2H)
6.7-7.3 (m, 6H)
1.5 (t, 3H)
―
8.3 (s, 2H)
H2L19
13.8 (s, 2H)
6.7-7.3 (m, 6H)
3.8 (m, 1H)
8.3 (s, 2H)
H2L20
13.1 (s, 2H)
6.8-7.3 (m, 10H)
1.4 (d, 3H) 1.5 (t, 3H) 1.5 (t, 3H)
4.0 (s, 4H) 4.1 (q, 2H) 3.9 (d, 2H) 4.2 (q, 2H)
―
8.6 (s, 2H)
H2L2 H2L3 H2L4
7.1-8.1(m,13H) 6.8-8.1(m, 13H) 6.9-8.0 (m, 14H)
138
Table 4.3.1 Selected infrared spectral bands of the Schiff bases and oxovanadium(IV) complexes Compounds
ν(C=N)
ν(C―O)
ν(V=O)
ν(V―N)
ν(V―O)
VOL1
1607, 1574
1363,1312
979
485, 537
573, 456
H2L1
1611, 1583
1313,1276
−
−
−
VOL2
1607, 1576
1362,1315
982
483, 541
581, 457
2
H2L
1621, 1567
1333,1289
−
−
−
VOL3
1604 ,1573
1361,1324
988
498, 540
572, 455
H2L3
1610, 1569
1316,1283
−
−
−
VOL4
1607, 1574
1366,1321
970
558, 536
488, 458
H2L4
1615, 1568
1313,1286
−
−
−
VOL5
1599
1337
986
495
406
H2L5
1604
1334
−
−
−
VOL6
1599
1336
989
519
489
H2L6
1607
1331
−
−
−
VOL7
1601
1337
959
528
418
7
H2L
1604
1331
−
−
−
VOL8
1606
1343
988
577
506
H2L8
1634
1284
−
−
−
VOL9
1618
1281
852
549
466
H2L9
1621
1257
−
−
−
VOL10
1600
1306
979
503
561
H2L10
1615
1287
−
−
−
VOL11
1616
1297
967
493
546
H2L11
1633
1278
−
−
−
VOL12
1627
1297
988
498
554
12
H2L
1631
1277
−
−
−
VOL13
1630
1309
860
471
516
H2L13
1636
1280
−
−
−
VOL14
1612
1322
969
435
524
H2L14
1615
1275
−
−
−
VOL15
1627
1277
979
491
573
139
H2L15
1638
1285
−
−
−
VOL16
1634
1307
848
476
549
H2L16
1640
1275
−
−
−
VOL17
1599
1289
981
497
544
H2L17
1622
1272
−
−
−
VOL18
1631
1313
976
483
611
H2L18
1637
1275
−
−
−
VOL19
1611
1304
982
471
614
H2L19
1631
1272
−
−
−
VOL20
1602
1315
982
448
541
H2L20
1614
1283
−
−
−
140
Table 4.3.2 Selected infrared spectral bands of cobalt(II), nickel(II) and copper(II) complexes Complex
ν(C=N)
ν(C−O)
ν(M−N)
ν(M−O)
CoL1
1604, 1578
1326, 1288
453, 508
575, 423
CoL2
1603, 1574
1363, 1319
454, 539
568, 428
CoL3
1606, 1577
1342, 1314
475, 548
568, 428
CoL4
1603, 1571
1337, 1313
580, 554
463, 428
NiL1
1609, 1582
1331, 1287
457, 554
577, 431
NiL2
1603, 1580
1366, 1311
457, 510
545, 431
NiL3
1606, 1577
1346, 1290
478, 507
560, 431
4
NiL
1606, 1577
1343, 1290
583, 507
463, 431
CuL1
1606, 1582
1325, 1290
457, 548
574, 419
CuL2
1606, 1580
1363, 1317
472, 501
563, 452
CuL3
1606, 1580
1340, 1311
498, 542
560, 463
CuL4
1609, 1577
1340, 1314
574, 545
460, 416
141
Table 4.4.1 Electronic spectral data of the cobalt(II) complexes Compound Solvent d-d transitions/cm-1 (ε/cm-1mol-1) CoL1
CHCl3
C.T
18 939 (77), 22 523 (205), 24 876 (273)
Ligand# 31 949 (228) 40 323 (606)
CoL2
DMSO
25 126 (236)
CHCl3
18 587 (59), 22 272 (153), 25 575 (227)
29 586 (183) 31 447 (202) 40 816 (364)
DMSO CoL3
CHCl3
25 063 (333)
36 101 (363)
18 868 (93), 22 523 (233), 24 876 (279) 28 902 (270) 40 323 (675) 30 675 (274
CoL4
DMSO
25 063 (228)
CHCl3
18 832 (94), 22 779 (250), 25 189 (337)
29 412 (238) 31 546 (277) 41 494 (668)
DMSO
25 445 (263)
#
29 586 215)
The spectra were noisy in the UV and some of the bands in the region could not be extracted.
142
Table 4.4.2 Electronic spectral data of the nickel(II) complexes C.T
Ligand#
20 619 (154), 25 840 (361)
30 675 (211)
38 314 (697)
DMSO
20 790 (145), 26 042 (322)
30 581 (184)
39 063 (658)
CHCl3
20 704 (127), 26 178 (312)
30 769 (204)
38 760 (437)
DMSO
20 877 (156), 26 178 (384)
Compound
Solvent
NiL1
CHCl3
NiL2
d-d transitions/cm-1 (ε/cm-1mol-1)
31 250 (216) 38 610 (501)
NiL3
NiL4
CHCl3
20 619 (162), 25 773 (383)
30 303 (234)
38 314 (700)
DMSO
20 619 (161), 25 974 (375)
30 395 (232)
38 168 (830)
CHCl3
20 747 (173), 25 840 (402)
30 675 (234)
38 314 (703)
DMSO
20 877 (150), 26 110 (340)
30 581 (191)
38 168 (638)
#
The spectra were noisy in the UV and some of the bands in the region could not be extracted.
143
Table 4.4.3 Electronic spectral data of the copper(II) complexes Compound
Solvent
CuL1
CHCl3
DMSO
d-d transitions/cm-1 (ε/cm-1mol-1) 23 041 (280)
23 256 (307)
C.T
Ligand#
27 778 (176)
31 056 (240)
29 326 (198)
37 736 (482)
30 030 (215)
31 646 (223) 37 313 (529)
CuL2
CHCl3
24 038 (184)
28 571 (213)
31 447 (225) 36 900 (344) 40 816 (371)
CuL3 CuL4
DMSO
23 981 (362)
36 101 (356)
CHCl3
22 831 (262)
29 674 (269)
37 736 (421)
DMSO
23 095 (262)
30 030 (252)
36 765 (408)
CHCl3
23 419 (308),
27 933 (203)
31 250 (262) 38 168 (438)
DMSO
23 585 (322),
28 490 (181), 30 395 (244)
#
The spectra were noisy in the UV and some of the bands in the region could not be extracted.
144
Table 4.4.4 Electronic spectral data of the oxovanaduim(IV) complexes Compound VOL1
Solvent CHCl3 DMSO
2
VOL
Band maxima/cm-1 (ε/cm-1mol-1) Band I
Band II
Band III
16 393
16 393
23 641(2 390)
(99)
(99)
16 129
16 129
(10)
(10)
CHCl3
C.T
Ligand#
27 701(1 380)*
31 447 (18 200) 40 323 (53 800)
23 753 (2,180)
31 746 (1 690)* 38 610 (1 550)
21 598*(2,375)
28 329 (860)
31 746 (890) 36 364 (1 220)* 40 323 (1 990)
DMSO
15 748
15 748 (9)*
27 473 (4,400)
(9) VOL3
CHCl3
16 207
31 746 (3 640) 36 101 (4 140)
16 207 (9)
23 256 (2 090)
28 736 (2 250)
41 322 (5 000)
15 898 (9)
23 474 (2,100)
29 155 (2,230)
38 610 (3 730)
16 234(20)
24 096 (2 270)
27 701(1 450)*
31 348 (1 860)
(9) DMSO
15 898 (9)
VOL4
CHCl3
16 234 (20)
DMSO 5
VOL
40 323 (5 000)
16 051
16 051
(10)
(10)
CHCl3
17 182
24 096 (2 080)
31 153 (1 700) 38 168 (3 220)
21 097 (82)*
27 473 (4 360)
(127)
35 461 (2 697) 40 486 (4 486) 41 152 (4 481)
DMSO VOL6
13 870
17 575
(461)
(484)
CHCl3
16 863
21 277 (453)*
27 322 (7 448)
20 747 (154)*
27 027 (1,002)
(150)
34 965 (2 563) 35 336 (2 364) 40 486 (3 973) 41 322 (4 051)
DMSO
17 123
21 276 (161)*
27 397 (970)
36 765 (2 355)*
21 186 (398)*
27 397 (714)
35 714 (1 929)
(205) CHCl3
12 255
16 026
145
VOL7
(94)
(24)
40 323 (3 849)
16 667
40 984 (3 857)
(226) DMSO VOL8
12 225
19 231
(37)*
(140)
CHCl3
16 155
21 097 (166)
27 624 (854)
25 840 (1 660)
29 851 (2 650)
(47) DMSO
16 269
41 322 (7 160) 24 213 (1 190)
(18) VOL9
CHCl3
36 900 (3 680)*
26 110 (1 780)
37 313 (5 080)
30 581 (2 600) 25 840 (1 490)
30 395 (2 580)
36 900 (3 680)* 41 322 (6 850)
DMSO VOL10
12 887
19 455
(6)
(11)
CHCl3
26 178 (1350)
32 154(2 520) 37 879(5 020)
15 942
21 749 (2 950)
(36)
23 474 (3 790)
28 169 (2 680)
31 746 (1 920) 36 765 (4 670) 40 650 (8 810)
DMSO
15 528
23 641(3 060)
(13)
21 786
28 736 (2 080)
36 630(4 180)
26 178(910)
35 714(2 350)*
(2 350)* VOL11
CHCl3
16,778 (25)
DMSO VOL12
CHCl3
39 683(5 220)
17 544 15)
26 882 (910)
13 263
16 892
26 178 (955)
(450)
(25)
35 971 (2 440)* 39 841 (5 193) 40 650 (6 234) 41 152 (4 420)
DMSO
12 970
17 699
(46)*
(14)
21 186 (549)
26 810 (893)
37 879(3 896) 38 610 (4 659) 38 911 (5 517)
VOL14
CHCl3
23 753 (1 140)
30 864 (1 410)
39 841 (2 520) 40 816 (2 580)
DMSO CHCl3
16 694
24 096 (18)
31 447 (1 950)
37 879 (2 750)
24 631 (10)
34 722(2 660)*
39 370 (4 210)
146
VOL15
(19) DMSO
16 891
25 380 (1090)
37 879 (4 270)
(17) VOL16
CHCl3 DMSO
VOL17
14 728
19 685
(76)
(90)
13 850
18 762
(19)
(30)
CHCl3 DMSO
24 876 (11)
35 336 (2950)*
25 445 (1 010)
37 879 (3 350)
22 422 (1 790)
31 348 (2 590)
39 370 (3 930)
22 624 (1 790)
31 646 (2 500)
38 023 (3 390)
25 510 (620)
33 333 (2 280)
40 984 (3 660)
17 065
26 385 (650)
38 760 (3 290)
(14)
34 364 (2 150)
15 649 (9)
VOL18
CHCl3
16 694 (15)
DMSO 19
VOL
CHCl3
16 667
25 316 (6)
33 445 (2 250)
40 816 (3 540)
26 110 (650)
34 130 (2 200)
(16) DMSO
16 694 (15)
VOL20
38 314 (2 760)
CHCl3
16 051 (7)
22 831 (1 180)
28 818 (3 040)
40 650 (3 550)
DMSO
16 155 (9)
23 641 (1 410)
30 395 (2 990)
37 879 (2 920)
#
The spectra were noisy in the UV and some of the bands in the region could not be extracted. * = Shoulder
147
Table 4.5 Room temperature magnetic moments for the complexes Complex
Magnetic moment/B.M.
Complex
Magnetic moment/B.M.
CoL1
2.61
VOL1
1.73
CoL2
2.59
VOL2
1.73
CoL3
2.23
VOL3
1.60
CoL4
2.36
VOL4
1.81
NiL1
diamagnetic
VOL5
1.74
NiL2
diamagnetic
VOL6
1.68
NiL3
diamagnetic
VOL7
1.78
NiL4
diamagnetic
VOL8
1.71
CuL1
2.20
VOL9
1.79
CuL2
1.56
VOL10
1.83
CuL3
1.81
VOL11
1.78
CuL4
1.78
VOL12
1.84
VOL13
1.76
VOL14
1.80
VOL15
1.72
VOL16
1.82
VOL17
1.83
VOL18
1.77
VOL19
1.72
VOL20
1.78
148
Table 4.6.1 EPR parameters for the oxovanadium(IV) complexes with axial symmetry Complexes Solution (290 K)
Solution (120 K)
giso
Aiso (G)
g⊥
g//
A// (G)
A⊥ (G)
VOL1
1.971
99.5
1.978
1.950
64
179.0
VOL2
1.971
101.0
1.978
1.950
65.0
179.0
VOL3
1.971
99.0
1.978
1.950
65.0
178.0
VOL4
1.971
99.5
1.978
1.950
64.0
177.0
VOL5
1.969
98.0
1.978
1.948
60.0
177.5
VOL6
1.969
98.0
1.978
1.949
60.0
177.0
VOL7
1.967
97.0
1.976
1.947
59.0
176.0
VOL9
1.970
96.5
1.972
1.950
58.0
170.0
VOL12
1.970
99.0
1.979
1.948
60.0
179.0
Table 4.6.2 EPR parameters for the oxovanadium(IV) complexes with rhombic symmetry Complexes
solution (290K)
solution (120K)
giso
Aiso (G)
gx
gy
gz
Ax (G)
Ay (G)
Az (G)
VOL10
1.972
99.0
1.976
1.980
1.950
63.0
58.0
178.0
VOL14
1.970
100.0
1.976
1.978
1.948
64.0
59.0
179.0
VOL19
1.97
99.0
1.976
1.978
1.948
64.0
60.0
179.0
149
Table 4.7 Cyclic voltammetric data for oxovanadium(IV) complexes Complexes Redox
Ep.c/mV
Ep.a/mV
ic/106
ia/106 ia/ic
E½/mV
ΔEp/mV
couple VOL1
VIV/VV
613
530
6.91
5.31
0.77
571.5
83
VOL
V
V /V
552
469
7.87
6.26
0.80
510.5
83
VOL4
VIV/VV
568
490
6.91
5.45
0.79
529
78
VOL5
VIV/VV
457
378
8.77
8.65
0.99
417.5
79
VOL6
VIV/VV
465
387
8.48
8.47
1.00
426
78
VOL7
VIV/VV
438
364
4.75
3.93
0.83
401
74
VOL8
VIV/VV
456
379
8.42
6.03
0.72
417.5
77
VOL9
VIV/VV
538
458
9.12
8.22
0.90
498
80
VOL10
VIV/VV
590
507
7.46
5.79
0.78
548.5
83
VOL11
VIV/VV
521
442
9.31
8.68
0.93
481.5
79
VOL12
VIV/VV
3
IV
525
444
10.09
9.39
0.93
484.5
81
VOL
V
V /V
521
440
10.15
9.07
0.89
480.5
81
VOL14
VIV/VV
662
580
7.90
5.46
0.69
621
82
VOL15
VIV/VV
380
297
1.07
1.02
0.95
338.5
83
VOL16
VIV/VV
490
414
8.92
8.45
0.95
452
76
VOL17
VIV/VV
512
428
8.96
7.27
0.81
470
84
VOL18
VIV/VV
395
317
9.21
8.81
0.96
356
78
VOL19
VIV/VV
399
319
8.21
7.93
0.97
359
80
VOL210
VIV/VV
528
448
8.22
6.45
0.78
488
80
13
IV
150
Table 4.8 DSC phenomenological data of oxovanadium(IV) complexes
complex
DSC peak temperature (o C) Endothermic peak
Exothermic peak
VOL1
350, 365,385
455
VOL2
―
380
VOL3
310
365
VOL4
335
455
VOL5
401
―
VOL7
345
―
VOL8
390
―
VOL9
330
―
VOL10
420
―
VOL11
420
430
12
VOL
315
360, 395
VOL13
415
425
VOL14
460
―
VOL15
325
440
VOL16
348
390
VOL17
345
415
VOL18
290
360
VOL19
240
360
VOL20
310
375
151
Table 5 Glucose uptake data for the oxovanadium(IV) complexes Compound
% glucose uptake
SD
DMSO
100.00
0.07
Insulin
185.70
0.28
0.010
Metformin
175.58
0.47
0.001
VOL1
154.16
0.34
0.007
2
VOL
142.58
0.39
0.001
VOL3
153.38
0.51
0.001
VOL4
167.34
0.72
0.009
VOL5
151.69
0.21
0.001
VOL6
93.84
0.17
-0.07
VOL7
166.77
0.58
0.001
VOL8
158.62
0.46
0.001
VOL9
111.78
0.19
0.2
VOL10
128.77
0.25
0.006
VOL11
155.20
0.36
0.001
VOL12
119.30
0.23
0.4
VOL13
156.04
0.62
0.001
VOL14
130.45
0.33
0.03
VOL15
200.04
0.54
0.01
VOL16
169.33
0.29
0.001
VOL17
179.70
0.36
0.008
VOL18
145.23
0.29
0.001
VOL19
107.24
0.21
0.3
VOL20
90.67
0.18
-0.04
152
p=value
APPENDIX II 1
0.9
0.8
0.7
%T
0.6
0.5
0.4
0.3
0.2
[Co(naph-Clsal)opd] H2(naph-Clsal)opd
0.1
0 2000
1800
1600
1400
1200
1000
800
600
400
Wavenumber (cm-1)
Fig. 4.3.1 IR spectra of complex CoL1 and its ligand H2(naph-Clsal)opd: representative spectra for the unsymmetrical complexes of cobalt(II) and the ligands 1
0.9
0.8
0.7
%T
0.6
0.5
0.4
0.3
0.2
[Ni(naph-sal)opd] H2(naph-sal)opd
0.1
0 2000
1800
1600
1400
1200
1000
800
600
400
Wavenumber (cm-1)
Fig. 4.3.2 IR spectra of complex NiL4 and its ligand H2(naph-Clsal)opd: representative spectra for the unsymmetrical complexes of nickel(II) and the ligands
153
1
0.9
0.8
0.7
%T
0.6
0.5
0.4
0.3 [Cu(naph-Clsal)opd]
0.2
H2(naph-Clsal)opd
0.1
0 2000
1800
1600
1400
1200
1000
800
600
400
Wavenumber (cm-1)
Fig. 4.3.3 IR spectra of complex CuL1 and its ligand H2(naph-Clsal)opd: representative spectra for the unsymmetrical complexes of copper(II) and the ligands
1
0.9
0.8
0.7
%T
0.6
0.5
0.4
0.3 VOL1
0.2
H2(naph-Clsal)opd
0.1
0 2000
1800
1600
1400
1200
1000
800
600
Wavenumber (cm-1)
Fig. 4.3.4 IR spectra of complex VOL1 and its ligand H2(naph-Clsal)opd: representative spectra for the unsymmetrical complexes of oxovanadium(IV) and the ligands
154
400
1 0.9 0.8 0.7
%T
0.6 0.5 0.4 0.3 0.2
VOL5 H2(bp)2en
0.1 0 2000
1800
1600
1400
1200
1000
800
600
400
Wavenumber (cm-1)
Fig. 4.3.5 IR spectra of complex VOL5 and its ligand H2bp2en: representative spectra for the benzophenoneimine
1
0.9
0.8
0.7
%T
0.6
0.5
0.4
0.3 VOL8
0.2
H2naph2en
0.1
0 2000
1800
1600
1400
1200
1000
800
600
400
Wavenumber (cm-1)
Fig. 4.3.6 IR spectra of complex VOL8 and its ligand H2naph2en: representative Spectra for the naphthaldiimine
155
1
0.9
0.8
0.7
%T
0.6
0.5
0.4
0.3
0.2
VOL13 H2(Clsal)2tn
0.1
0 2000
1800
1600
1400
1200
1000
800
600
400
Wavenumber (cm-1)
Fig. 4.3.7 IR spectra of complex VOL13 and its ligand H2Clsal2tn: representative spectra for the chlorosalicylaldiimine
1
0.9
0.8
0.7
%T
0.6
0.5
0.4
0.3 VOL15 H2Omesal2en
0.2
0.1
0 2000
1800
1600
1400
1200
1000
800
600
400
Wavenumber (cm-1)
Fig. 4.3.8 IR spectra of complex VOL15 and its ligand H2Omesal2en: representative spectra for the methoxysalicylaldiimine
156
1
0.9
0.8
0.7
%T
0.6
0.5
0.4
0.3 VOL18
0.2
H2Oetsal2en
0.1
0 2000
1800
1600
1400
1200
1000
800
600
400
Wavenumber (cm-1)
Fig. 4.3.9 IR spectra of complex VOL18 and its ligand H2Oetsal2en: representative spectra for the ethoxysalicylaldiimine
157
0.7
A
0.6
Absorbance
0.5 CoL1 CoL2 CoL3 CoL4
0.4
0.3
0.2
0.1
0 250
300
350
400
450
500
550
600
Wavelength (nm)
0.6
B 0.5
Absorbance
0.4 CoL1 CoL2 CoL3 CoL4
0.3
0.2
0.1
0 250
300
350
400
450
500
550
600
Wavelength (nm1)
A: [complexes] = 10-5 in CHCl3; B: [complexes] = 10-5 in DMSO Fig. 4.4.1 Electronic spectra of the unsymmetrical cobalt(II) complexes
158
0.8
A
0.7
0.6
NiL1 NiL2 NiL3 NiL4
Absorbance
0.5
0.4
0.3
0.2
0.1
0 250
300
350
400
450
500
550
600
Wavelength (nm)
0.8
B 0.7
0.6 NiL1 NiL2 NiL3 NiL4
Absorbance
0.5
0.4
0.3
0.2
0.1
0 250
300
350
400
450
500
550
600
Wavelength (cm-1)
A: [complexes] = 10-5 in CHCl3; B: [complexes] = 10-5 in DMSO Fig. 4.4.2 Electronic spectra of the unsymmetrical nickel(II) complexes
159
0.6
A 0.5
Absorbance
0.4 CuL1 CuL2 CuL3 CuL4
0.3
0.2
0.1
0 250
300
350
400
450
500
550
600
Wavelength (nm)
0.6
B 0.5
Absorbance
0.4
CuL1 CuL2 CuL3 CuL4
0.3
0.2
0.1
0 250
300
350
400
450
500
550
600
Wavelength (nm1)
A: [complexes] = 10-5 in CHCl3; B: [complexes] = 10-5 in DMSO Fig. 4.4.3 Electronic spectra of the unsymmetrical copper(II) complexes
160
1.2
A
1.1 1 0.9
Absorbance
0.8 VOL1 VOL2 VOL3 VOL4
0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 500
550
600
650
700
750
800
Wavelength (nm)
0.4
B
Absorbance
0.3
VOL1 VOL2 VOL3 VOL4
0.2
0.1
0 500
550
600
650
700
750
800
Wavelength (nm)
A: [complexes] = 10-3 in CHCl3; B: [complexes] = 10-3 in DMSO Fig. 4.4.4 Electronic spectra of the unsymmetrical oxovanadium(IV) complexes
161
0.4
A
VOL1 VOL2 VOL3 VOL4
Absorbance
0.3
0.2
0.1
0 250
300
350
400
450
500
Wavelength (nm)
0.5
B 0.4 VOL1 VOL2 VOL3 VOL4 Absorbance
0.3
0.2
0.1
0 250
300
350
400
450
Wavelength (nm)
A: [complexes] = 10-5 in CHCl3; B: [complexes] = 10-5 in DMSO Fig. 4.4.5 Electronic spectra of the unsymmetrical oxovanadium(IV) complexes
162
500
0.5
A VOL5
0.4
VOL6 VOL7
Absorbance
0.3
0.2
0.1
0 500
550
600
650
700
750
800
Wavelength (nm)
0.6
B 0.5
Absorbance
0.4
VOL5
0.3
VOL6 VOL7
0.2
0.1
0 500
550
600
650
700
750
800
Wavelength (nm)
A: [complexes] = 10-3 in CHCl3; B: [complexes] = 10-3 in DMSO Fig. 4.4.6 Electronic spectra of the benzophenoneiminatooxovanadium(IV) complexes
163
0.5
A 0.4 VOL5 VOL6 VOL7
Absorbance
0.3
0.2
0.1
0 250
300
350
400
450
500
Wavelength (nm)
0.3
B VOL5 VOL6 VOL7
Absorbance
0.2
0.1
0 250
300
350
400
450
500
Wavelength (nm)
A: [complexes] = 10-5 in CHCl3; B: [complexes] = 10-5 in DMSO Fig. 4.4.7 Electronic spectra of the benzophenoneiminatooxovanadium(IV) complexes
164
0.7
A 0.6 VOL8
Absorbance
0.5
0.4
0.3
0.2
0.1
0 500
550
600
650
700
750
800
Wavelength (nm)
0.5
B 0.4 VOL8 VOL9 VOL10
Absorbance
0.3
0.2
0.1
0 500
550
600
650
700
750
800
Wavelength (nm)
A: [complexes] = 10-3 in CHCl3; B: [complexes] = 10-3 in DMSO Fig. 4.4.8 Electronic spectra of the napthaldimiinatooxovanadium(IV) complexes
165
0.9
A
0.8
0.7 VOL8 VOL9
Absorbance
0.6
0.5
0.4
0.3
0.2
0.1
0 250
300
350
400
450
500
Wavelength (nm)
0.9
B
0.8
0.7 VOL8 VOL9 VOL10
Absorbance
0.6
0.5
0.4
0.3
0.2
0.1
0 250
300
350
400
450
500
Wavelength (nm)
A: [complexes] = 10-5 in CHCl3; B: [complexes] = 10-5 in DMSO Fig. 4.4.9 Electronic spectra of the napthaldiiminatooxovanadium(IV) complexes
166
0.5
A 0.4
VOL11 VOL12
Absorbance
0.3
0.2
0.1
0 500
550
600
650
700
750
800
Wavelenght (nm)
2
B VOL11 VOL12 VOL13
Absorbance
1.5
1
0.5
0 500
550
600
650
700
750
800
Wavelength (nm)
A: [complexes] = 10-3 in CHCl3; B: [complexes] = 10-3 in DMSO Fig. 4.4.10 Electronic spectra of the chlorosalicylaldiiminatooxovanadium(IV) complexes
167
0.7
A 0.6
Absorbance
0.5 VOL11 VOL12 VOL13
0.4
0.3
0.2
0.1
0 250
300
350
400
450
500
Wavelength (nm)
0.7
B 0.6
Absorbance
0.5 VOL11 VOL12 VOL13 VOL14
0.4
0.3
0.2
0.1
0 250
300
350
400
450
500
Wavelength (nm)
A: [complexes] = 10-5 in CHCl3; B: [complexes] = 10-5 in DMSO Fig. 4.4.11 Electronic spectra of the chlorosalicylaldiiminatooxovanadium(IV) complexes
168
2
A VOL15 VOL16 VOL17
Absorbance
1.5
1
0.5
0 500
550
600
650
700
750
800
Wavelength (nm)
0.5
B 0.4 VOL15 VOL16 VOL17
Absorbance
0.3
0.2
0.1
0 500
550
600
650
700
750
800
Wavelength (nm)
A: [complexes] = 10-3 in CHCl3; B: [complexes] = 10-3 in DMSO Fig. 4.4.12 Electronic spectra of the methoxysalicylaldiiminatooxovanadium(IV) complexes
169
0.5
A 0.4
VOL15 VOL16 VOL17
Absorbance
0.3
0.2
0.1
0 250
300
350
400
450
500
Wavelength (nm)
0.5
B 0.4
VOL15 VOL16 VOL17
Absorbance
0.3
0.2
0.1
0 250
300
350
400
450
500
Wavelength (nm)
A: [complexes] = 10-5 in CHCl3; B: [complexes] = 10-5 in DMSO Fig. 4.4.13 Electronic spectra of the methoxysalicylaldiiminatooxovanadium(IV) complexes
170
0.3
A
VOL18 VOL19 VOL20
Absorbance
0.2
0.1
0 500
550
600
650
700
750
800
Wavelength (nm)
0.3
B
VOL18 VOL19 VOL20
Absorbance
0.2
0.1
0 500
550
600
650
700
750
800
Wavelenght (nm)
A: [complexes] = 10-3 in CHCl3; B: [complexes] = 10-3 in DMSO Fig. 4.4.14 Electronic spectra of the ethoxysalicylaldiiminatooxovanadium(IV) complexes
171
0.3
A
0.2
Absorbance
VOL18 VOL19 VOL20
0.1
0 250
300
350
400
450
500
Wavelength (nm)
0.5
B 0.4
VOL18 VOL19 VOL20
Absorbance
0.3
0.2
0.1
0 250
300
350
400
450
500
Wavelength (nm)
A: [complexes] = 10-5 in CHCl3; B: [complexes] = 10-5 in DMSO Fig. 4.4.15 Electronic spectra of the ethoxysalicylaldiiminatooxovanadium(IV) complexes
172
- experimental - simulation
1
2800
3000
3200
3400
3600
3800
- experimental - simulation
4000
2500
Gauss
3000
3500
2
- experimental - simulation
2800
3000
3200
3400
4000
Gauss
3600
3800
- experimental - simulation
4000
Gauss
2500
3000
3500
4000
Gauss
3 - experimental - simulation
2800
3000
3200
3400
3600
3800
- experimental - simulation
4000
Gauss
2500
3000
4
3500
4000
Gauss - experimental - simulation
2800
3000
3200
3400
3600
3800
- experimental - simulation
4000
2500
Gauss
3000
3500
4000
Gauss
Fig. 4.5.1 EPR spectra of complexes 1 [VOL1], 2 [VOL2], 3 [VOL3] and 4 [VOL4] at 290K (left) and 120 K (right) in toluene/CH2Cl2
173
2800
- experimental - simulation
- experimental - simulation
5
3000
3200
3400
3600
3800
4000
2500
3000
6
2800
3000
3200
3400
3600
3800
4000
2500
3000
3200
3400
3600
3800
4000
2500
3000
3500
- experimental - simulation
3200
3400
4000
Gauss
8
3000
4000
- experimental - simulation
- experimental - simulation
Gauss
2800
3500
Gauss
7
3000
4000
- experimental - simulation
- experimental - simulation
Gauss
2800
3500
Gauss
Gauss
3600
3800
4000
- experimental - simulation
2500
3000
3500
4000
Gauss
Gauss
Fig. 4.5.2 EPR spectra of complexes 5 [VOL5], 6[VOL6], 7 [VOL7] and 8 [VOL12] at 290K (left) and 120 K (right) in toluene/CH2Cl2
174
VOL3
VOL8
0.000006
0 .0 0 0 0 0 8 0 .0 0 0 0 0 6
0.000004
0 .0 0 0 0 0 4
0.000002
0 .0 0 0 0 0 2
Current/µA
Current/µA
0.000000 -0.000002 -0.000004
0 .0 0 0 0 0 0 -0 .0 0 0 0 0 2 -0 .0 0 0 0 0 4 -0 .0 0 0 0 0 6
-0.000006
-0 .0 0 0 0 0 8 -0 .0 0 0 0 1 0
-0.000008
-0 .0 0 0 0 1 2
-0.000010
-0 .2
-0.2
0.0
0.2
0.4
0.6
0.8
0 .0
0 .2
0 .4
0 .6
0 .8
1 .0
P o te n tia l/V
1.0
P otential/V
VOL5
VOL11
0.000008
0.000008
0.000006
0.000006
0.000004
0.000004 0.000002
0.000000
Current/µA
Current/µA
0.000002
-0.000002 -0.000004 -0.000006
0.000000 -0.000002 -0.000004 -0.000006
-0.000008
-0.000008
-0.000010 -0.2
0.0
0.2
0.4
0.6
0.8
1.0
-0.000010
Potential/V
-0.2
0.0
0.2
0.4
0.6
0.8
1.
Potential/V
VOL15
VOL18
0.000008
0.000008
0.000006
0.000006
0.000004
0.000004
0.000002
0.000002
Current/µA
Current/µA
0.000000 -0.000002 -0.000004 -0.000006
0.000000 -0.000002 -0.000004 -0.000006
-0.000008
-0.000008
-0.000010
-0.000010
-0.000012 -0.2
0.0
0.2
0.4
0.6
0.8
1.0
-0.000012 -0.2
Potential/V
0.0
0.2
0.4
Potential/V
Fig. 4.6 Cyclic voltammograph of the oxovanadium(IV) complexes: Representative voltammograph for each of the series
175
0.6
0.8
1.0
DSC /(mW/mg) ↑ exo
Temp. /°C [1]
[1] VS18.dd5
450
DSC Temp.
-0.5
400 -1.0 [1]
-1.5
350
300
250
-2.0
200 -2.5 150 -3.0 100
50
-3.5
0
5
10
Time /min
15
20
Fig. 4.7.1.1 DSC curves of VOL1
DSC /(mW/mg) ↑ exo
Temp. /°C [1]
[1] VS15.dd5
450
DSC Temp.
30
400 25 350
20 300
250
15
200 10 150 5 100
50
0 [1]
0
5
10
Time /min
15
Fig. 4.7.1.2. DSC curves of VOL2
176
20
DSC /(mW/mg) ↑ exo
Temp. /°C [1]
[1] VS16.dd5
450
DSC Temp.
-1.0
400 -1.5 350 -2.0 300 -2.5 250 -3.0 200 -3.5 150 -4.0 100 -4.5 50 [1]
0
5
10
Time /min
15
20
Fig. 4.7.1.3. DSC curves of VOL3
DSC /(mW/mg) ↑ exo -0.5
Temp. /°C [1]
[1] VS17.dd5
450
DSC Temp. -1.0
400 -1.5 350 -2.0 300
-2.5 250
-3.0 [1]
200
-3.5 150
-4.0
100
-4.5
50
0
5
10
Time /min
15
Fig. 4.7.1.4. DSC curves of VOL4
177
20
DSC /(mW/mg) ↑ exo
Temp. /°C [1]
[1] VS3.dd5
450
DSC Temp.
-1
400 -2 [1]
350 -3 300 -4 250 -5 200 -6 150 -7 100 -8 50 -9 0
5
10
Time /min
15
20
Fig. 4.7.2.1.1. DSC curves of VOL5
DSC /(mW/mg) ↑ exo
Temp. /°C [1]
[1] VS5.dd5
450
DSC Temp.
-0.5
400 -1.0 350
-1.5
300
-2.0
250
200
-2.5
150 -3.0 [1]
100
-3.5 50 -4.0 0
5
10
Time /min
15
Fig. 4.7.2.1.3. DSC curves of VOL7
178
20
DSC /(mW/mg) ↑ exo
Temp. /°C
[1] vs27.dd5
[1]
DSC Temp.
0.0
450
-0.2
400
-0.4
350 [1]
300
-0.6
250
-0.8
200
-1.0
150 -1.2 100 -1.4 50 -1.6 0
5
10
15
Time /min
20
Fig. 4.7.2.2.1. DSC curves of VOL8
DSC /(mW/mg) ↑ exo
Temp. /°C [1]
[1] VS12.dd5
450
DSC Temp.
-1
400 -2
350 -3
[1]
300 -4
250
-5
200
-6
150
-7
100
-8
50
-9
0
5
10
Time /min
15
Fig. 4.7.2.2.2. DSC curves of VOL9
179
20
DSC /(mW/mg) ↑ exo
Temp. /°C [1]
[1] VS11.dd5
450
DSC Temp. -1
400
-2
350
[1]
300
-3
250 -4 200
-5
150
100
-6
50 -7 0
5
10
Time /min
15
20
Fig. 4.7.2.2.3. DSC curves of VOL10
DSC /(mW/mg) ↑ exo 0.5
Temp. /°C [1]
[1] VS25.dd5
450
DSC Temp. 0.0
400 -0.5 350 -1.0 300 -1.5
[1]
250 -2.0 200 -2.5 150 -3.0 100 -3.5 50 -4.0 0
5
10
Time /min
15
Fig. 4.7.2.3.1. DSC curves of VOL11
180
20
DSC /(mW/mg) ↑ exo
Temp. /°C [1]
[1] VS1.dd5
450
DSC Temp. -1
400
350
-2
300
[1]
250 -3 200
150 -4 100
50 -5 0
5
10
Time /min
15
20
Fig. 4.7.2.3.2. DSC curves of VOL12
DSC /(mW/mg) ↑ exo
Temp. /°C [1]
[1] VS2.dd5
8
450
DSC Temp.
6
400
4
350
2
300
0 250 -2 200 [1]
-4
150 -6 100 -8 50 -10 0
5
10
Time /min
15
Fig. 4.7.2.3.3. DSC curves of VOL13
181
20
DSC /(mW/mg) ↑ exo
Temp. /°C [1]
[1] VS21.dd5
450
DSC Temp.
-0.5
400 -1.0 350 -1.5 300 [1]
-2.0
250 -2.5 200 -3.0 150 -3.5 100 -4.0 50 -4.5 0
5
10
Time /min
15
20
Fig. 4.7.2.3.4. DSC curves of VOL14
DSC /(mW/mg) ↑ exo
Temp. /°C [1]
[1] VS24.dd5
450
DSC Temp.
-1
400 -2
[1]
350 -3 300 -4 250 -5 200 -6 150 -7 100 -8 50 -9 0
5
10
Time /min
15
Fig. 4.7.2.4.1. DSC curves of VOL15
182
20
DSC /(mW/mg) ↑ exo
Temp. /°C
[1] vs19.dd5
[1]
DSC Temp.
[1]
450
0
400 -1 350 -2 300
250
-3
200 -4 150 -5 100
50
-6
0
5
10
15
Time /min
20
Fig. 4.7.2.4.2. DSC curves of VOL16
DSC /(mW/mg) ↑ exo
Temp. /°C [1]
[1] VS22.dd5
450
DSC Temp. 0
400
-1 350
-2
300
[1]
-3
250
200 -4 150 -5 100
-6
50
0
5
10
Time /min
15
Fig. 4.7.2.4.3. DSC curves of VOL17
183
20
DSC /(mW/mg) ↑ exo
Temp. /°C [1]
[1] VS26.dd5
450
DSC Temp.
0.0
400 -0.5 350
-1.0
300
-1.5
-2.0
[1]
250
-2.5
200
-3.0
150
-3.5
100
-4.0
-4.5
50
0
5
10
15
Time /min
20
Fig. 4.7.2.5.1. DSC curves of VOL18
DSC /(mW/mg) ↑ exo
Temp. /°C [1]
[1] VS23.dd5
450
DSC Temp. -1
400
350 -2 300
[1]
250
-3 200
150 -4 100
50
-5
0
5
10
Time /min
15
Fig. 4.7.2.5.2. DSC curves of VOL19
184
20
DSC /(mW/mg) ↑ exo 4
Temp. /°C [1]
[1] VS20.dd5
450
DSC Temp.
400 2 350
0
300
250 -2 200
[1]
-4
150
100 -6 50
0
5
10
Time /min
15
Fig. 4.7.2.5.3. DSC curves of VOL20
185
20
250.00
*
**
200.00
*
*
**
**
% glucose uptake
**
**
**
150.00
Series1
100.00
50.00
0 L1 VO
L9 VO
L8 VO
L7 VO
L6 VO
L5 VO
L4 VO
L3 VO
L1
L2 VO
fo M
et
VO
rm
in
l in su In
DM
SO
0.00
Fig. 5.1.1 Glucose uptake from the culture media containing 8mM glucose by C2C12 cells over one 1 h. C2C12 cells were pre-exposed to the complexes, insulin and metformin respectively in glucose and serum free media for 3 h before the glucose uptake experiments. Basal glucose uptake, i.e. solvent vehicle only (DMSO), is represented as 100% and the subsequent increase or decrease induced by the compounds is reflected as ± 100%. Statistical significances of p≤0.01 and 0.001 are indicated by * and ** respectively.
186
250.00
*
* **
*
200.00
*
**
**
**
% g lu co se u ptake
150.00 Series1
100.00
50.00
0 VO
L2
9 L1 VO
VO
L1
8
7 L1 VO
VO
L1
6
5 L1 VO
VO
L1
4
3 L1 VO
VO
L1
2
1 L1
rm fo et M
VO
in
l in su In
DM
SO
0.00
Fig. 5.1.2 Glucose uptake from the culture media containing 8mM glucose by C2C12 cells over one 1 h. C2C12 cells were pre-exposed to the complexes, insulin and metformin respectively in glucose and serum free media for 3 h before the glucose uptake experiments. Basal glucose uptake, i.e. solvent vehicle only (DMSO), is represented as 100% and the subsequent increase or decrease induced by the compounds is reflected as ± 100%. Statistical significances of p≤0.01 and 0.001 are indicated by * and ** respectively.
187
20
% R ed u ctio n
10 0 -10 -20
*
-30
** *
-40 1
2
Control
VOL1 0.2 mmol
3
4
Hours treatment VOL2 after 0.2 mmol VOL3 0.2 mmol
5
6
VOL4 0.2 mmol
Fig. 5.2.1 The effect of the unsymmetrical complexes on hyperglycemia in Wistar outbred rats with STZ-induced diabetes is shown. A bar-graph with SEM error bars represents the hourly percentage changes in the whole blood glucose values over the 6-hour monitoring period.The percentage changes in the plasma glucose values of the treatment groups in the STZ rat model were calculated for each hour and then subtracted from the percentage changes in the control values (normalized against the control) at each time-point. A statistical significance of p≤0.05 is indicated by *, statistical significance 0.01 – 0.0001 is indicated by ** and statistical significance p ≤ 0.0001 is indicated by ***.
188
30 20 % R ed u ctio n
10 0 -10
*
-20 -30
**
-40
*
-50 1
Control
2
VO5 0.2 mM/kg
3
4
Hours after VO6 0.2 mM/kg VO7treatment 0.2 mM/kg
5
VO12 0.2 mM/kg
6
VO13 0.2 mM/kg
Fig. 5.2.2 The effect of five symmetrical complexes on hyperglycemia in Wistar outbred rats with STZ-induced diabetes is shown. A bar-graph with SEM error bars represents the hourly percentage changes in the whole blood glucose values over the 6-hour monitoring period.The percentage changes in the plasma glucose values of the treatment groups in the STZ rat model were calculated for each hour and then subtracted from the percentage changes in the control values (normalized against the control) at each time-point. A statistical significance of p≤0.05 is indicated by *, statistical significance 0.01 – 0.0001 is indicated by ** and statistical significance p ≤ 0.0001 is indicated by ***.
189